Transportin-1: A Nuclear Import Receptor with Moonlighting Functions Allegra Mboukou, Vinod Rajendra, Renata Kleinova, Carine Tisné, Michael Jantsch, Pierre Barraud

To cite this version:

Allegra Mboukou, Vinod Rajendra, Renata Kleinova, Carine Tisné, Michael Jantsch, et al.. Transportin-1: A Nuclear Import Receptor with Moonlighting Functions. Frontiers in Molecular Biosciences, Frontiers Media, 2021, 8, pp.638149. ￿10.3389/fmolb.2021.638149￿. ￿hal-03171125￿

HAL Id: hal-03171125 https://hal.archives-ouvertes.fr/hal-03171125 Submitted on 16 Mar 2021

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. REVIEW published: 18 February 2021 doi: 10.3389/fmolb.2021.638149

Transportin-1: A Nuclear Import Receptor with Moonlighting Functions

Allegra Mboukou 1, Vinod Rajendra 2, Renata Kleinova 2, Carine Tisné 1, Michael F. Jantsch 2 and Pierre Barraud 1*

1Expression Génétique Microbienne, Institut de Biologie Physico-Chimique (IBPC), UMR 8261, CNRS, Université de Paris, Paris, France, 2Department of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria

Transportin-1 (Trn1), also known as karyopherin-β2 (Kapβ2), is probably the best- characterized nuclear import receptor of the karyopherin-β family after Importin-β, but certain aspects of its functions in cells are still puzzling or are just recently emerging. Since the initial identification of Trn1 as the nuclear import receptor of hnRNP A1 ∼25 years ago, several molecular and structural studies have unveiled and refined our understanding of Trn1-mediated nuclear import. In particular, the understanding at a molecular level of the NLS recognition by Trn1 made a decisive step forward with the identification of a new class of NLSs called PY-NLSs, which constitute the best-characterized substrates of Trn1. Besides PY-NLSs, many Trn1 cargoes harbour NLSs that do not resemble the archetypical PY-NLS, which complicates the global understanding of cargo recognition by Trn1. Although PY-NLS recognition is well established and supported by several Edited by: structures, the recognition of non-PY-NLSs by Trn1 is far less understood, but recent Maria Rosaria Conte, King’s College London, reports have started to shed light on the recognition of this type of NLSs. Aside from its United Kingdom principal and long-established activity as a nuclear import receptor, Trn1 was shown more Reviewed by: recently to moonlight outside nuclear import. Trn1 has for instance been caught in Tobias Madl, Medical University of Graz, Austria participating in virus uncoating, ciliary transport and in modulating the phase Roberta Spadaccini, separation properties of aggregation-prone proteins. Here, we focus on the structural University of Sannio, Italy and functional aspects of Trn1-mediated nuclear import, as well as on the moonlighting *Correspondence: activities of Trn1. Pierre Barraud [email protected] Keywords: karyopherin, karyopherin-β2, nucleo-cytoplasmic transport, NLS, proline–tyrosine nuclear localization signal, phase-separation, FUS, TNPO1 Specialty section: This article was submitted to Structural Biology, 1 INTRODUCTION a section of the journal Frontiers in Molecular Biosciences In eukaryotic cells, the presence of a physical separation between the nucleus and the cytoplasm in Received: 05 December 2020 the form of a double membrane assures a physical and temporal separation of the transcription and Accepted: 13 January 2021 translation processes but creates the need for a selective and efficient transport of thousands of Published: 18 February 2021 macromolecules across the nuclear envelope (Görlich and Kutay, 1999; Conti and Izaurralde, 2001; Citation: Fried and Kutay, 2003; Cook et al., 2007). This continual ballet, with controlled bidirectional flows, is Mboukou A, Rajendra V, Kleinova R, orchestrated by nuclear transport receptors (NTRs) that carry their cargoes from one compartment Tisné C, Jantsch MF and Barraud P (2021) Transportin-1: A Nuclear Import to the other by crossing the nuclear envelope at the level of the nuclear pore complexes (NPCs) (Tran Receptor with Moonlighting Functions. and Wente, 2006; Wente and Rout, 2010; Allegretti et al., 2020). Transport receptors of the Front. Mol. Biosci. 8:638149. karyopherin-β (Kapβ) family account for the vast majority of the cargo flow through the NPC. doi: 10.3389/fmolb.2021.638149 Karyopherins interact selectively with proteins of the NPC, namely the phenylalanine-glycine

Frontiers in Molecular Biosciences | www.frontiersin.org1 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting nucleoporins (FG-Nups), which surround and line the NPC transport receptors. Trn1 was in fact later shown to only central channel (Hampoelz et al., 2019). Within the NPC, function in nuclear import, with the hnRNP A1 cargo being these FG-Nups built and establish the proper operation of the released from Trn1 upon RanGTP binding (Siomi et al., 1997; permeability barrier (Fu et al., 2018). In general, large Izaurralde et al., 1997), a common trait of nuclear import macromolecules (>40 kDa) are indeed excluded from the NPC receptors, but the terminology has remained. channel, whereas karyopherins can cross the NPC barrier on In the Kapβ family, Trn1 shares the highest sequence identity account of their common property to selectively interact with FG- (83%) with Transportin-2 (Trn2), also known as karyopherin- Nups (Rout et al., 2003; Weis, 2007; Hülsmann et al., 2012; Beck β2B (Kapβ2B) (Twyffels et al., 2014). Trn2 exists in two isoforms and Hurt, 2017). called Trn2A or Kapβ2B(A) (Siomi et al., 1997) and Trn2B or Karyopherins that mediate nuclear import are also known as Kapβ2B(B) (Shamsher et al., 2002), the expression of which importins, whereas those mediating nuclear export are known as results from an alternative splicing event (Rebane et al., 2004). exportins. Besides the binding to FG-Nups, importins and This high sequence similarity between Trn1 and Trn2 correlates exportins associate with their cargoes via signals, namely with a highly similar cargo spectrum for these two NTRs. Among nuclear localization signals (NLSs) and nuclear export signals different karyopherins, Trn1 indeed presents the highest degree (NESs), which determine whether the cargo is imported in or of functional redundancy with Trn2 in terms of cargoe specificity exported out of the nucleus (Chook and Süel, 2011; Xu et al., (Mackmull et al., 2017; Kimura et al., 2017). Their cargoes include 2010; Cook and Conti, 2010). Importins bind to their cargoes in many RNA-binding proteins involved in mRNA processing, such the cytoplasm, reach the NPCs and translocate to the other side of as proteins of the hnRNP family (e.g., hnRNP A1, hnRNP A2/B1, the nuclear envelope, where they release their cargoes upon hnRNP D, hnRNP F, hnRNP H, hnRNP M), but also other binding to the small GTPase Ran in its GTP-bound form protein families implicated in nucleic-acid-related functions in (RanGTP). In a reciprocal manner, exportins associated with the nucleus. Although the exclusive import of specific cargoes by RanGTP bind to their cargoes in the nucleus, reach the NPCs and either Trn1 or Trn2 have not been experimentally confirmed, a translocate to the other side of the nuclear envelope, where they high-throughput study suggested a slight specialization of these dissociate from their cargoes upon GTP hydrolysis and RanGDP NTRs. Cargoes with efficient NLSs would be imported by any of release (Conti and Izaurralde, 2001; Fried and Kutay, 2003; Weis, the two Transportins, whereas cargoes with less efficient NLSs 2003). Transport directionality is thus primarily driven by the might be exclusively carried by either Trn1 or Trn2 (Kimura et al., RanGTPase nucleotide cycle, which produces an asymmetric 2017). For instance, actin and actin-related proteins implicated in distribution of RanGTP and RanGDP on both sides of the chromatin remodeling and transcription, and proteins related to nuclear envelope, with RanGTP being present at high nuclear division, would preferentially be imported by Trn1, concentrations in the nucleus and with RanGDP being mainly whereas proteins related to DNA repair would preferentially present in the cytoplasm (Izaurralde et al., 1997; Dasso, 2002). be Trn2 cargoes (Kimura et al., 2017). These recent studies Transport selectivity, on the other hand, relies on the selective aiming at defining the cargo spectrum of individual NTRs binding of NTRs to their cargoes via the selective recognition of clearly provide insightful information on NTR-cargo their signals. recognition. However, these studies must be analyzed with Within the Kapβ family, Transportin-1 (Trn1), also known as caution, since among the few hundreds of potential Trn1 karyopherin-β2 (Kapβ2), is probably the best-characterized cargoes identified in two recent reports, only ∼20 are common nuclear import receptor after Importin-β [also known as to the two datasets (Mackmull et al., 2017; Kimura et al., 2017; karyopherin-β1 (Kapβ1)]. Despite nearly identical structures, Baade and Kehlenbach, 2019). The need for experimental Trn1 shows only 24% sequence similarity to Importin-β validation would therefore be essential to understand in detail (Cingolani et al., 1999; Chook and Blobel, 1999). They share a the cargo-specificity of Trn1 and to establish the potential higher similarity at the level of their N-terminal half where the specialization of Trn1 and Trn2. binding of RanGTP takes place, and differ deeply at the level of Aside from its principal and long-established activity as a their C-terminal half, the site of binding to their respective nuclear import receptor, Trn1 was shown more recently to cargoes. Whereas Importin-β binds via the adaptor protein moonlight outside nuclear import. Trn1 has been for instance Importin-α to cargoes harbouring the ‘classical’ NLS (cNLS), caught in participating in virus uncoating, cellular cilia formation Trn1 binds directly to its cargoes without adaptor (Chook and and in modulating the phase separation properties of Süel, 2011; Cook et al., 2007; Twyffels et al., 2014). Trn1 has been aggregation-prone proteins. In this review, we will focus on initially identified as the nuclear import receptor of the the structural and functional aspects of Trn1-mediated nuclear heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) import, as well as on the moonlighting activities of Trn1. (Pollard et al., 1996; Fridell et al., 1997; Bonifaci et al., 1997). The NLS sequence of hnRNP A1 [designated the ‘M9’ sequence (Siomi and Dreyfuss, 1995; Weighardt et al., 1995)] can also 2 TRANSPORTIN-1: A NUCLEAR IMPORT mediate its nuclear export (Michael et al., 1995). Therefore, RECEPTOR considering that the newly identified NTR could both transport hnRNP A1 in and out of the nucleus, this 2.1 Structural Organization of Trn1 karyopherin was named ‘transportin’ in opposition to the Like other Kapβ, Trn1 is composed of HEAT-repeats and adopts names ‘importins’ and ‘exportins’ in use for unidirectional an overall superhelical architecture (Figure 1). HEAT-repeats

Frontiers in Molecular Biosciences | www.frontiersin.org2 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

FIGURE 1 | Structural organization of Transportin-1. (A) Trn1 is composed of 20 HEAT repeats and adopts a superhelical architecture [free Trn1: PDB code 2QMR (Cansizoglu and Chook, 2007)]. Helices of the HEAT repeats are displayed as cylinders in gray. The loops connecting the different helices are in blue, except for the loop of HEAT H14 that is in green and for the long acidic loop of HEAT H8 (H8 loop) that is in red. Most of the H8 loop is unstructured in the free Trn1 structure, and this long loop is schematically represented with a dashed line. Trn1 structure is overall formed by two consecutive and overlapping arches. The N-terminal arch consists of HEATs H1-H13, and the C-terminal arch is formed by HEATs H8-H20. (B) RanGTP (in yellow) associates with Trn1 (in gray) and fits snugly into the N-terminal arch [Trn1- RanGTP complex: PDB code 1QBK (Chook and Blobel, 1999)]. Upon binding, the H8 loop (in red) is reorganized and becomes almost entirely structured. (C) The hnRNP A1 ‘M9’ NLS (in purple) associates with Trn1 (in gray) in an extended conformation that covers almost entirely the C-terminal arch [Trn1-NLS complex: PDB code 2H4M (Lee et al., 2006)]. As in free Trn1, most of the H8 loop is unstructured in the NLS-bound structure of Trn1. (D) The three major segments identified to describe the conformational flexibility of Trn1 are depicted with three distinct colors, namely HEATs H1-H8 in red, H9-H13 in orange, and H14-H20 in yellow. These segments are displayed on Trn1 surface using two 180°-views to help better grasp the superhelical character of this HEAT-repeat protein.

consist of tandem repeats of a ∼40-residue motif (Andrade et al., helix B via a small loop of ∼2–4 residues, except at the level of 2001)thatwasfirst identified in Huntingtin, Elongation factor 3, HEAT H14, where this loop is slightly longer and consists of 9 the regulatory A subunit of protein phosphatase 2A and the residues, and notably also at the level of HEAT H8, where the two TOR lipid kinase (Andrade and Bork, 1995). Each HEAT motif helices of the motif are connected via a long loop of about sixty folds into a pair of α-helices (known as the A and B helices). residues (Figure 1A). This long loop, thereafter called the H8 Consecutive HEAT motifs pile together in a nearly parallel loop, is intrinsically disordered and has an overall negative charge fashion, which lead to an overall superhelical architecture. (27 Asp/Glu over 65 residues). A longer H8 loop is also present in The inner concave surface of Trn1 is formed by the B other Kapβ, such as Importin-β, but it is significantly longer in helices, whereas the A helices are exposed at the outer Trn1 (65 vs. 12 residues in Importin-β)(Cingolani et al., 1999; convex surface. The different structures of Trn1, either free Chook and Blobel, 1999; Cook et al., 2007). or in complex with its different partners, namely RanGTP or Structural and functional considerations have led to define variousNLSs(seeTable 1), all showed that Trn1 is composed of Trn1 as formed by two consecutive and overlapping arches. The 20 HEAT repeats. N-terminal arch consists of HEATs H1-H13, and the C-terminal As in other Kapβ proteins, the HEAT motifs in Trn1 are arch is formed by HEATs H8-H20 (Figure 1A). The N-terminal connected to each other via small loops or small helices. In arch is the site of interaction with RanGTP (Chook and Blobel, addition, within each HEAT motif, helix A is connected to 1999), whereas the C-terminal arch is the site of NLS recognition,

Frontiers in Molecular Biosciences | www.frontiersin.org3 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

TABLE 1 | Crystal structures of Transportin-1.

Transportin-1 Partner Type of NLS PDB code References

Trn1-FL RanGTP 1QBK (Chook and Blobel, 1999) Trn1-ΔH8 hnRNP A1 NLS (‘M9’ NLS) hPY-NLS 2H4M (Lee et al., 2006) Trn1-ΔH8 hnRNP M NLS bPY-NLS 2OT8 (Cansizoglu et al., 2007) Trn1-FL – 2QMR (Cansizoglu and Chook, 2007) Trn1-FL – 2Z5J (Imasaki et al., 2007) Trn1-FL TAP/NXF1 NLS PY-NLS 2Z5K, 2Z5M (Imasaki et al., 2007) Trn1-FL hnRNP D NLS bPY-NLS 2Z5N (Imasaki et al., 2007) Trn1-FL hnRNP DL/JKTBP NLS PY-NLS 2Z5O (Imasaki et al., 2007) Trn1-ΔH8 FUS NLS hPY-NLS 4FDD (Zhang and Chook, 2012) Trn1-FL FUS NLS hPY-NLS 4FQ3 (Niu et al., 2012) Trn1-ΔH8 ScNab2 NLS ∼PY-NLS 4JLQ (Soniat et al., 2013) Trn1-ΔH8 HCC1 NLS PY-NLS 4OO6 – Trn1-ΔH8 histone H3 NLS non-PY-NLS 5J3V (Soniat and Chook, 2016) Trn1-ΔH8 RPL4 PY-NLS 5TQC (Huber and Hoelz, 2017) Trn1-ΔH8 FUS NLS hPY-NLS 5YVG, 5YVH, 5YVI (Yoshizawa et al., 2018)

Trn1-FL: construct of Trn1 full-length. Trn1-ΔH8: construct of Trn1 with a shortened H8 loop. hPY-NLS: hydrophobic PY-NLS. bPY-NLS: basic PY-NLS. PY-NLS: PY-NLS without a basic or hydrophobic stretch seen to interact with Trn1 in the structure. ∼PY-NLS: variation to the PY-NLS motif.

as seen for the ‘M9’ sequence of hnRNP A1 (Lee et al., 2006) spring-like effect. In the different structures (Table 1), Trn1 is (Figures 1B,C). more stretched or more compact, and this occurs mostly at the Conformational flexibility is a hallmark of the Kapβ family of level of its N- and C-terminal flexible repeats (H1-H4 and H19- proteins (Conti et al., 2006). In Trn1, this intrinsic structural H20). This gives an overall structure that can be more or less flexibility, which presumably allows Trn1 to adapt its extended. In the structures and the dynamic simulation, free Trn1 conformation for binding its different partners, namely FG- is the one adopting the most elongated conformation, whereas the Nups, RanGTP and various NLSs, has been initially RanGTP- and the ‘M9’-NLS-bound forms are more compact investigated at low-resolution using small-angle X-ray (Cansizoglu and Chook, 2007; Wang et al., 2012). It is worth scattering (SAXS) (Fukuhara et al., 2004). Later, with a noting that longer loops connecting helices A and B are found at growing number of Trn1 structures (Table 1), either free the level of the three main segment bounds, namely the H8 loop (Cansizoglu and Chook, 2007), or in complex with RanGTP and the H14 loop. These two loops and the surrounding regions (Chook and Blobel, 1999) and the ‘M9’ NLS of hnRNP A1 (Lee constitute dynamic ‘hotspots’ important for Trn1 function et al., 2006), the conformational flexibility of Trn1 has been (Wang et al., 2012), namely cargo binding and its release upon analyzed at atomic-resolution using structure coordinates and binding to RanGTP (see below). some atomic-refinement parameters (namely the crystallographic B-factors) of free Trn1 (Cansizoglu and Chook, 2007). In 2.2 Trn1-Mediated Import Pathway addition, the conformational flexibility of Trn1 has been The first step of the Trn1 import cycle consists in the recognition analyzed in silico using molecular dynamics simulations and binding of the NLS of a Trn1 cargo in the cytoplasm (Wang et al., 2012). Together, these studies have shown that (Figure 2, step 1). NLS recognition by Trn1 will be described the behaviour in terms of conformational flexibility is not in detail in the following subsections, but overall, binding occurs uniform along the Trn1 structure, and that substructures or in the C-terminal arch of Trn1, as seen for the ‘M9’ sequence of segments can be distinguished for describing the overall hnRNP A1 (Lee et al., 2006)(Figure 1C). In the cytoplasm, conformational flexibility of Trn1. The conformational RanGTP is scarce and binding of Trn1 to its cargoes occurs in its flexibility of Trn1 has therefore been reported as ‘segmental’, free form. with three major segments composed of HEATs H1-H8, H9-H13, Then, Trn1 associated with its cargo via the NLS transits and H14-H20 (Figure 1D). The two latest segments are relatively towards the NPCs, where its biochemical properties and ability to rigid substructures that rotate around a flexible hinge at the level interact with FG-Nups allows it to pass through the NPC of H13-H14 helices. The first segment is intrinsically more selectivity barrier (Figure 2, step 2). heterogeneous and can be subdivided into two smaller In the nucleus, RanGTP is abundant, and cargo-associated segments, the segment H1-H4, which is highly prone to Trn1 binds to RanGTP, which triggers cargo release (Figure 2, conformation changes, and the segment H5-H8, the step 3). RanGTP binds Trn1 at the concave surface of the conformation of which is mostly sensitive to RanGTP binding N-terminal arch, and more precisely at the level of the (Cansizoglu and Chook, 2007; Wang et al., 2012). Within the segments H1-H4 and H7-H8 but also at the level of the long segment H14-H20, repeats H19-H20 are the most prone to and acidic H8 loop (Chook and Blobel, 1999)(Figure 1B). Parts conformational changes. of the ‘switch I’ and ‘switch II’ regions of Ran (switch I: residues The conformational flexibility of Trn1 is conferred by the 30–47; switch II: residues 65–80), which experience large repetition of the HEAT motifs, the packing of which produces a conformational changes during the interconversion between

Frontiers in Molecular Biosciences | www.frontiersin.org4 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

FIGURE 2 | Nuclear import cycle of Transportin-1. (step 1) Trn1 associates with its cargo via the NLS in the cytoplasm [free Trn1 in gray: PDB code 2QMR (Cansizoglu and Chook, 2007); hnRNP A1 cargo with its unstructured ‘M9’ NLS in purple: PDB code 2LYV (Barraud and Allain, 2013); Trn1-cargo in gray and purple: PDB code 2H4M (Lee et al., 2006)]. (step 2) Trn1 associated with its cargo transits towards and passes through the NPC. The NPC, with the nuclear basket and the cytoplasmic filaments, is schematically represented in green. (step 3) Trn1 associated with its cargo binds to RanGTP in the nucleus, which triggers cargo release [RanGTP in yellow: PDB code 1RRP (Vetter et al., 1999); Trn1-RanGTP in gray and yellow: PDB code 1QBK (Chook and Blobel, 1999)]. (step 4) Trn1 associated with RanGTP transits towards and passes through the NPC. (step 5) At the cytoplasmic face of the NPC, RanGTP is hydrolyzed and released from Trn1 upon the combined action of RanBPs and RanGAP (RanBP1 in green and RanGAP in red: PDB code 1K5D (Seewald et al., 2002); RanGDP in blue: PDB code 1BYU (Stewart et al., 1998)]. Trn1 is free again in the cytoplasm and ready for another import cycle. the different nucleotide states of Ran (Vetter et al., 1999; Blobel, 1999). The part of the H8 loop that does not make Scheffzek et al., 1995; Cook et al., 2007), are recognized at the direct contact with RanGTP adopts an extended conformation level of the H1-H3 segment of Trn1. Similarly to what is observed and interacts extensively with the segment H12-H18 in the in Impβ, the basic patch of Ran (residues 139–142), located C-terminal arch (Figure 1B). The H8 loop thereby occupies opposite to the switch regions of Ran, is involved in the NLS interaction sites as observed for the ‘M9’ sequence of electrostatic interactions with HEAT repeat H7 in Trn1, but hnRNP A1 (Figure 1C). The interaction of the loop with the not directly with the acidic H8 loop (Lee et al., 2005). NLS-binding site depends on RanGTP, and the displacement and Although the RanGTP-bound structure of Trn1 is less structuring of the H8 loop at the NLS-binding site provides a elongated than the free Trn1 structure, the associated molecular mechanism to the cargo-dissociation upon RanGTP structural change is only minor. In contrast, binding of binding (Lee et al., 2006; Chook and Blobel, 1999). The RanGTP to Trn1 has pronounced and extensive effects on the involvement of the H8 loop in cargo dissociation is also conformation of the H8 loop (Figures 1A,B). The long H8 loop, supported by the fact that its removal allows Trn1 to bind the which is almost entirely disordered in the free Trn1 structure, ‘M9’ NLS and RanGTP simultaneously (Chook et al., 2002). By becomes indeed well-structured upon interacting with RanGTP. investigating the binding and dissociation of several NLSs, the About one third of the H8 loop residues make direct contacts with mechanism of substrate release from Trn1 has been refined and RanGTP. These contacts include both hydrophobic interactions was reported to occur in a stepwise manner. Upon RanGTP with apolar side-chains and aromatics, and electrostatic binding, NLS dissociation would first occur at the level of the interactions with polar and charged side-chains (Chook and segment H14-H18, also called ‘site B’, and complete release of the

Frontiers in Molecular Biosciences | www.frontiersin.org5 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting cargo would then occur upon dissociation of the NLS from the pioneering work, several studies have refined the segment H8-H13, also called ‘site A’ (Imasaki et al., 2007). understanding of PY-NLS recognition by Trn1 (see subsection Then, Trn1 associated with RanGTP transits towards the 3.1). However, many Trn1 cargoes harbour NLSs that do not NPCs, and interacts with FG-Nups to pass through the NPC resemble PY-NLSs (Chook and Süel, 2011; Twyffels et al., 2014). selectivity barrier, similarly to its entry into the nucleus, but in the The recognition of these non-PY-NLSs by Trn1 is until now far opposite direction (Figure 2, step 4). less understood than that of PY-NLSs. Whether non-PY-NLSs When Trn1 bound to RanGTP reaches the cytoplasmic face of can be regarded as variations around the PY-NLS paradigm, or the NPC, it associates with several cytoplasmic factors involved in whether they are too divergent and use completely different RanGTP hydrolysis and dissociation from the karyopherin, recognition rules, has not been clearly established yet, mostly namely the Ran GTPase-activating protein (RanGAP) and the by lack of studies, including structural ones, focused on non-PY- Ran-binding proteins RanBP1 and RanBP2. RanBP2, also known NLSs (see subsection 3.2). Finally, over the years, a salient aspect as nucleoporin 358 (Nup358), harbors four Ran-binding domains of Trn1-mediated nuclear import has emerged and consists in the (RanBD) and is a component of the cytoplasmic filaments of the many reported mechanisms that modulate the karyopherin-cargo NPC (Delphin et al., 1997). The intrinsic GTP-hydrolysis by Ran interactions, thereby enabling nuclear import regulation (see is extremely slow, and RanGAP is needed in the cytoplasm to subsection 3.3). stimulate the hydrolysis rate by several orders of magnitude (Bischoff et al., 1994; Klebe et al., 1995; Cook et al., 2007). 3.1 PY-NLS Recognition However this enhancement is drastically dampened when Unlike the classical-NLSs recognized by the Impα/Impβ system, RanGTP is associated with karyopherins (Floer and Blobel, PY-NLSs cannot be described solely by a traditional consensus 1996; Bischoff and Görlich, 1997; Fried and Kutay, 2003), and sequence. They are instead described by a collection of loose RanGAP-mediated stimulation of GTP-hydrolysis necessitates principles that can be summarized as follows: a peptide segment the implication of RanBPs, which destabilize the RanGTP/ of 15–30 residues with intrinsic structural disorder, an overall karyopherin complex. RanGTP hydrolysis therefore occurs at basic character, and some weakly conserved sequence motifs, the cytoplasmic face of the NPC and involves the combined including a relatively conserved proline–tyrosine (PY) dipeptide, action of RanBPs and RanGAP (Figure 2, step 5). Upon GTP which gave the name PY-NLS to this class of signals (Lee et al., hydrolysis, Ran switches conformation, both at the level of the 2006). More precisely, the sequence motifs of PY-NLSs are ‘switch I’ and ‘switch II’ regions, but also at the level of its composed of an N-terminal basic or hydrophobic motif C-terminal extension that folds as an α-helix and packs against followed by a C-terminal R/K/H-X2–5-P-Y motif (Lee et al., the Ran structural core (Vetter et al., 1999; Scheffzek et al., 1995). 2006). The nature of the N-terminal motif further divides PY- Altogether, these structural rearrangements provide a steric NLSs into basic and hydrophobic subclasses (bPY-NLSs and barrier that prevents RanGDP from binding back to the hPY-NLSs, respectively). In bPY-NLSs, the N-terminal motif karyopherin. After RanGTP release from Trn1 and GTP consists of a patch of basic residues, whereas in hPY-NLSs, the hydrolyis, Trn1 is again free in the cytoplasm and ready for equivalent N-terminal motif conforms to the loose Φ–G/A/ another import cycle (Figure 2, step 1). S–Φ–Φ consensus (where Φ is a hydrophobic residue). To date, ∼10 distinct crystal structures of Trn1 have been solved in complex with PY-NLS substrates, including both bPY- 3 NUCLEAR LOCALIZATION SIGNAL NLSs and hPY-NLSs (Table 1). Collectively, these structures RECOGNITION BY TRANSPORTIN-1 show that all PY-NLSs interact with the same region of Trn1, namely an extended zone of the concave face of the C-terminal In the years following the identification of Trn1 as the nuclear arch (Figure 3A). However, considering the poor resemblance of import receptor of hnRNP A1 (Pollard et al., 1996; Fridell et al., the NLSs in term of sequence, it is not surprising to see that 1997; Bonifaci et al., 1997), several other proteins (including structural superposition of the different PY-NLSs is really far hnRNP D, hnRNP F, hnRNP M, HuR, Y-box binding protein 1, from perfect (Figure 3B). PY-NLSs vary both in length and TAP, and histones H2A, H2B, H3 and H4) have been identified as sequence, and their binding mode to Trn1 may at first sight look Trn1 cargoes (Siomi et al., 1997; Fan and Steitz, 1998; Truant very disparate. However, structural convergence clearly appears et al., 1999; Mühlhäusser et al., 2001; Rebane et al., 2004; at two sites in the C-terminal part of the NLSs, and to a lesser Güttinger et al., 2004; Bader and Vogt, 2005; Suzuki et al., extent at one site in the N-terminal part. These three sites of 2005). However, the lack of sequence similarity between the structural convergence form three binding epitopes, which have different NLSs has for a long time impeded the identification been initially identified by the structural comparison of the of the important elements defining NLSs recognized by Trn1. The binding mode of hnRNP A1 and hnRNP M PY-NLSs understanding at a molecular level of the NLS recognition by (Cansizoglu et al., 2007), and were later shown to provide a Trn1 made a decisive step forward with the resolution of the first significant energetic contribution to the binding (Süel et al., structure of Trn1 in complex with an NLS, namely the ‘M9’ NLS 2008). Epitope 1, which shows the least structural convergence of hnRNP A1 (Figure 1C)(Lee et al., 2006). This work indeed and the highest sequence divergence, corresponds to the provided the means to identify common patterns among N-terminal motif presented above and can be formed by a seemingly contrasting NLSs and to group and classify them patch of either basic or hydrophobic residues. Epitope 2 and 3 into a new class of NLSs called PY-NLSs. Following this correspond to the positively charged residue and to the PY

Frontiers in Molecular Biosciences | www.frontiersin.org6 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

FIGURE 3 | PY-NLS recognition by Transportin-1. (A) A common binding site for PY-NLSs with the C-terminal arch of Trn1. Trn1 is shown in gray as a transparent surface to facilitate PY-NLS observation. The PY-NLSs are displayed with different colors (see below), and all share the same N-ter to C-ter orientation. (B) Structural superposition of PY-NLSs from the following proteins: hnRNP A1 in purple [PDB code 2H4M (Lee et al., 2006)], FUS in light blue [PDB code 4FDD (Zhang and Chook, 2012)], hnRNP M in green [PDB code 2OT8 (Cansizoglu et al., 2007)], hnRNP D in dark blue [PDB code 2Z5N (Imasaki et al., 2007)], TAP/NXF1 in red [PDB code 2Z5K (Imasaki et al., 2007)], HCC1 in orange (PDB code 4OO6), RPL4 in gray [PDB code 5TQC (Huber and Hoelz, 2017)], ScNab2 in yellow [PDB code 4JLQ (Soniat et al., 2013)]. The structures were superimposed on the Trn1 proteins, but for clarity they are not shown, and only the PY-NLSs are displayed as sticks. The three recognition epitopes are shown with colored ellipses. Epitope 1, which corresponds to a stretch of basic or hydrophobic residues, is highlighted in yellow. Epitope 2, which corresponds to a positively charged residue (R/K/H), is highlighted in green. Epitope 3, which corresponds to a proline–tyrosine dipeptide (PY) is highlighted in blue. (C) Structure-based alignment of the same PY-NLSs displayed on panel B. The PY-NLS sequences were aligned according to their relative position in the structures. Only residues actually observed in the structures are displayed. The three epitopes as described in panel B are highlighted with the same color code. Within epitope 1, hydrophobic residues are colored in blue, and positively charged residues are colored in red. hPY-NLS: hydrophobic PY-NLS. bPY-NLS: basic PY-NLS. PY- NLS: PY-NLS without a basic or hydrophobic stretch seen to interact with Trn1 in the structure. ∼PY-NLS: variation to the PY-NLS motif.

dipeptide of the C-terminal R/K/H-X2–5-P-Y motif, respectively interacting at both site A and site B, or uniquely at site A for (Figure 3B,C). the ones only interacting at the level of epitopes 2 and 3 (Imasaki What emerges from such structural superposition, is that there et al., 2007). Since the conformation of site A is independent of are clearly two types of PY-NLSs: the ones that are seen to interact NLS binding, whereas that of site B is intrinsically more with Trn1 all along the three epitopes (e.g., hnRNP A1, FUS, adaptable, it has been proposed that the C-terminal R/K/ fi hnRNP M and hnRNP D) and those that are only interacting at H-X2–5-P-Y motif would bind rst to site A, then a epitopes 2 and 3 (e.g., TAP/NXF1, HCC1, RPL4 and ScNab2). To conformational change of site B would occur owing to a help describe these two categories of PY-NLSs, and in connection rearrangement at the level of the H13-H14 hinge, which with with the structural heterogeneity and the segmental nature of a sort of induced fit mechanism would adapt site B to epitope 1 Trn1 (Figure 1D), two main NLS-binding sites have been defined binding (Imasaki et al., 2007). As previously mentioned, substrate within the Trn1 C-terminal arch. The first one, called ‘site A’, dissociation upon RanGTP binding is likely occurring in the corresponds to HEATs H8-H13, and represents the site of reverse order, with PY-NLSs dissociating first from site B, and interaction of epitopes 2 and 3 (Figure 3C). The second one, then from site A (Imasaki et al., 2007). Molecular mechanisms called ‘site B’, corresponds to HEATs H14-H20, and is the site of governing proper functioning of Trn1 (e.g., substrate binding and interaction of epitope 1 (Figure 3C). PY-NLSs are thus release) are therefore deeply linked to its structural adaptability.

Frontiers in Molecular Biosciences | www.frontiersin.org7 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

It is important to mention that although the structures of PY- NLSs, but uses a strong epitope 1 that compensates for the loss of NLSs in complex with Trn1 do not always show an interaction at the PY motif. The non-PY-NLS of histone H3 can thus be seen as the level of site B (Figures 3B,C), residues upstream of the R/K/ an ultimate variation of the PY-NLS, lacking the PY motif, but fi H-X2–5-P-Y motif seen to interact at site A can signi cantly retaining the other hallmarks of this class of NLSs. Whether this contribute to the overall binding (Imasaki et al., 2007; Süel et al., aspect is unique to histone H3, or is shared by other non-PY- 2008). For that reason, PY-NLSs shown to only interact at site A NLSs of Trn1, is a likely possibility, but remains to be determined may still be classified as bPY-NLSs or hPY-NLSs depending on by further studies, including structural works dealing with other their amino-acid composition (Lee et al., 2006; Twyffels et al., non-PY-NLSs. 2014). Most importantly, the binding site accommodating Although no other crystal structure of non-PY-NLSs with epitope 1 seems to be highly tolerant, since it can bind to both Trn1 has been obtained, molecular information on non-PY-NLS basic and hydrophobic patches, and a combination of both basic interactions with Trn1 are available for several systems. These and hydrophobic residues is often observed in the N-terminal include the FOXO4 (forkhead box O4) protein, which binds Trn1 motif (Figure 3C)(Lee et al., 2006). Overall, the distribution of using a very unusual mechanism (Putker et al., 2013). Upon the binding energy along the three binding epitopes highly accumulation of reactive oxidative species (ROS), a disulfide bond depends on the PY-NLS considered. In some cases, the is formed between a cysteine residue of FOXO4 and a cysteine contribution of epitope 3 is pre-eminent, whereas in others residue in Trn1. The formation of a covalent complex ensures a main binding contributions are provided by epitopes 1 and 2 strong interaction with Trn1 and an efficient nuclear import. In (Süel et al., 2008; Lee et al., 2006; Imasaki et al., 2007; Cansizoglu the nucleus, the more reducing environment facilitates the et al., 2007). This property, together with the fact that the epitopes reduction of the intermolecular disulfide bond and the release are energetically quasi-independent, had been proposed as of the FOXO4 transcription factor, which eventually activates responsible for the high adaptability of the epitopes, which can transcription of ROS-detoxifying enzymes. Neither the accommodate large sequence diversity on condition that the interaction site in Trn1, nor the implicated cysteine, have so others are energetically strong. A combinatorial mixing of far been identified. Residues surrounding the implicated cysteine energetically weak and strong motifs indeed maintains an in FOXO4 have been shown to participate in the binding to Trn1 overall affinity compatible with nuclear import (Süel et al., 2008). (Putker et al., 2013), but it remains to be determined whether this interaction occurs specifically at a single site on Trn1 surface or at 3.2 Non-PY-NLS Recognition several positions. Among other things, it would be interesting to In addition to the import of PY-NLS-containing cargoes, Trn1 is know whether the present binding site is completely different implicated in the import of various cargoes obviously lacking a from the one used by PY-NLSs or if they have some parts in PY-NLS (Twyffels et al., 2014; Chook and Süel, 2011). These common. cargoes are imported by Trn1 via non-PY-NLSs, and include A non-PY-NLS for which molecular details of its recognition diverse proteins such as the core histones (H2A, H2B, H3 and by Trn1 is available consists of the atypical NLS of the RNA H4) (Mühlhäusser et al., 2001; Baake et al., 2001; Mosammaparast editing enzyme ADAR1 (Eckmann et al., 2001; Strehblow et al., et al., 2002; Blackwell et al., 2007; Soniat et al., 2016), ribosomal 2002; Fritz et al., 2009). This NLS overlaps the third double- proteins (RPL23A, RPL5, RPL7 and RPS7) (Jäkel and Görlich, stranded RNA-binding domain (dsRBD) of the protein (Barraud 1998; Tai et al., 2013), viral proteins (Arnold et al., 2006; Le Roux and Allain, 2012), and shows no similarity to PY-NLSs. The and Moroianu, 2003; Klucevsek et al., 2006), the RNA-editing molecular basis for the dsRBD-mediated nuclear import of enzyme ADAR1 (Fritz et al., 2009; Barraud et al., 2014; Banerjee ADAR1 was investigated at a molecular level. The solution and Barraud, 2014), the transcription factor FOXO4 (Putker structure of the ADAR1-dsRBD3 revealed an extended dsRBD et al., 2013), and the cold-inducible RNA-binding protein fold with an additional α-helix in the N-terminus. This extension CIRBP (Bourgeois et al., 2020). With some exceptions, PY- radically changes the relative position of the flexible fragments NLS seem to be specific to Trn1, while non-PY-NLS can be flanking the dsRBD and brings the N- and C-terminal flanking imported by Trn1 but can frequently be recognized and imported regions in close proximity (Barraud et al., 2014). The two by multiple karyopherins such as the heterodimer Impα/Impβ fragments flanking the folded dsRBD were shown to constitute (Kimura et al., 2013; Twyffels et al., 2014). two essential modules involved in the interaction with Trn1 and Structural understanding of non-PY-NLS recognition by Trn1 the non-PY-NLS of ADAR1 was thus called ‘bimodular NLS’. The is quite limited, since only a single structure of Trn1 in complex intervening dsRBD was shown to only act as a scaffolding with a non-PY-NLS peptide has been solved to date (Soniat and domain, which properly positions the N- and C-terminal Chook, 2016). In this structure, the non-PY-NLS of histone H3 modules for an effective interaction with Trn1. Functional spans HEATs H11-H18 in the concave site of the bimodular NLSs could indeed be designed by replacing the Trn1 C-terminal arch. It occupies similar positions as the ones ADAR1-dsRBD3 with an unrelated dsRBD, or even with a of PY-NLSs bound to Trn1, with the exception that there is no PY small peptide linker, which clearly indicates that the dsRBD dipeptide or other residues occupying the epitope 3 recognition only helps bring together the two NLS-modules that are site of PY-NLSs (Figure 3). A positively charged stretch binds the otherwise distantly spaced in the protein sequence (Barraud site for PY-NLS epitope 1, and an arginine residue occupies the et al., 2014). Molecular modelling and functional assays PY-NLS epitope 2 position (Soniat and Chook, 2016). The involving Trn1 mutants affected in the regular PY-NLS binding mode is therefore somewhat similar to the one of PY- binding sites (Figure 3), namely ‘site A’ and ‘site B’, suggested

Frontiers in Molecular Biosciences | www.frontiersin.org8 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting that the bimodular NLS of ADAR1 may interact with Trn1 at the interaction with Trn1. It is worth noting that arginine same interaction sites as the ones of PY-NLSs. The folded dsRBD methylation is not the only mechanism that modulates would be small enough to insert into the C-terminal arch of Trn1, interaction of PABP-2 with Trn1, since binding of PABP-2 to and the N- and C-terminal modules of the non-PY-NLS of poly-A RNA competes with binding to Trn1. The two ADAR1 could adopt an extended conformation that allows mechanisms are interconnected since arginine methylation interaction with Trn1 at the position of epitopes 1–3, even slightly enhances binding of PABP-2 to RNA, which altogether though they do not contain a PY dipeptide (Barraud et al., reduces its interaction with Trn1 (Fronz et al., 2011). In cells, 2014). However, the atomic details of the interaction still whether this competition occurs in the cytoplasm or in the remain to be uncovered, which would expand our nucleus has not yet been firmly established. Similarly, asymmetric understanding of the repertoire of non-PY-NLS recognition arginine dimethylation of FUS was reported to reduce interaction by Trn1. with Trn1 thereby affecting nuclear import (Dormann et al., 2012). In another recent report, a non-PY-NLS has been identified in In contrast to the PABP-2 situation, arginine methylation does not the cold-inducible RNA-binding protein CIRBP and was shown occur directly within the PY-NLS of FUS but next to it, in the so- to participate in the Trn1-mediated nuclear import of CIRBP called RGG3 region present just upstream of the C-terminal PY- (Bourgeois et al., 2020). This non-PY-NLS corresponds to a ∼40- NLS. Interestingly, although arginine dimethylation modulates Trn1 residue region rich in RG and RGG motifs, called the RG/RGG binding to wild-type FUS with a fully functional PY-NLS, the effect region. This RG/RGG region binds to Trn1 in a RanGTP- of arginine methylation in the RGG3 region is more pronounced in competitive manner and does not contain PY or PΦ motifs the case of FUS mutants with defective PY-NLSs, and inhibition of (Φ: hydrophobic residue) that may be reminiscent of PY- arginine methylation in these mutant proteins restores an efficient NLSs. The most striking point regarding the RG/RGG region nuclear import (Dormann et al., 2012). Strikingly, it was later interaction with Trn1 consists in the involvement of the Trn1 H8 reported that in contrast to arginine dimethylation that drastically loop in the interaction (Bourgeois et al., 2020). The RG/RGG reduces the interaction of the RGG3 region of FUS with Trn1, region of CIRBP was indeed shown to contact Trn1 at two key arginine monomethylation only slightly affects this interaction and sites, a site competing with the binding of the PY-NLS of FUS, behaves similarly to unmethylated arginine in FUS (Suárez-Calvet and a non-overlapping site within the unstructured H8 loop of et al., 2016). Owing to their almost identical domain organization, Trn1. The importance of this additional site is reflected in the fact theFETproteins,namelyFUS,EWSandTAF15,aresimilarly that deletion of the H8 loop reduces the binding affinity of the affected by arginine methylation in their interaction properties with RG/RGG region to Trn1 by ∼5 fold (Bourgeois et al., 2020). As Trn1 (Dormann et al., 2012; Suárez-Calvet et al., 2016). Along the presented above, although the H8 loop has been shown to be same lines, arginine methylation of the non-PY-NLS of CIRBP, also essential for cargo release upon RanGTP binding, it was reported referred to as the RG/RGG region, reduces its binding affinity to as dispensable for NLS binding, and was indeed deleted in most Trn1, thereby regulating nuclear import of CIRBP (Bourgeois et al., structures of Trn1 in complex with PY-NLSs (Table 1). This 2020). study concerning the RG/RGG NLS of CIRBP raises the Lysine acetylation has been reported to regulate the interaction possibility that the Trn1 H8 loop might be important for NLS of FUS with Trn1. A lysine acetylation within the PY-NLS of FUS binding, at least for certain non-PY-NLSs (Bourgeois et al., 2020). was shown to disrupt the interaction between FUS and Trn1, To determine whether the disordered H8 loop is important for resulting in the reduction of FUS nuclear import and hence its the recognition of other non-PY-NLSs is definitely a critical mislocalization in the cytoplasm (Arenas et al., 2020). The question that would necessitate further studies. implicated lysine is situated in epitope 1 of FUS PY-NLS (Figure 3), and is directly in contact with a glutamate and an 3.3 Regulation of Trn1-Cargo Interactions aspartate residue of Trn1 HEATs H14 and H15 (Zhang and Several mechanisms modulating Trn1-cargo interactions, thereby Chook, 2012; Niu et al., 2012). The reduction of interaction with enabling nuclear import regulation, have been reported in the Trn1 upon acetylation of this specific lysine is thereby clearly in literature. This mostly includes the modulation of Trn1-cargo accordance with structural data. interactions via post-translational modifications (e.g., arginine Phosphorylations have been reported as modulator of Trn1- methylation, lysine acetylation, and serine or tyrosine cargo interactions in several contexts. First, the Trn1-mediated phosphorylation), but also the enhancement or inhibition of import of Sam68 was reported as regulated through the interaction by more elaborated mechanisms. phosphorylation (Lukong et al., 2005). The BRK kinase indeed phosphorylates Sam68 on three tyrosine residues in its PY-NLS, 3.1.1 Post-Translational Modifications Regulating including Y440 that forms the PY motif of PY-NLS epitope 3 Trn1-Cargo Interactions (Figure 3). While unphosphorylated Sam68 is predominantly Arginine methylation has been reported as a modulator of Trn1- nuclear, Sam68 phosphorylated on Y440 relocalizes to cargo interactions in several contexts. First, asymmetric arginine cytoplasmic perinuclear structures (Lukong et al., 2005), most dimethylation within the PY-NLS of the nuclear Polyadenylate- probably as a consequence of an impaired interaction with Trn1. binding protein 2 (PABP-2) reduces its affinity for Trn1 (Fronz Similarly, the FET proteins FUS and EWS have been shown to be et al., 2011). Six arginine residues that are likely involved in Trn1- phosphorylated on their C-terminal tyrosine (i.e., Y526 and Y656, binding as part of PY-NLS epitopes 1 and 2, are subjected to post- respectively), which forms the PY motif of their PY-NLSs translational modifications, which would explain the reduction of (Leemann-Zakaryan et al., 2011; Darovic et al., 2015). In the

Frontiers in Molecular Biosciences | www.frontiersin.org9 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

case of FUS, this phosphorylation completely abolishes the PY- et al., 2013). Unexpectedly, increased binding upon H2O2 NLS interaction with Trn1 (Darovic et al., 2015). The tyrosine treatment did not lead to an increased nuclear import of residue that is phosphorylated forms epitope 3 of FUS PY-NLS PER1, but was rather associated with a specific slow-down of (Figure 3), and contacts several Trn1 residues, including an PER1 nuclear import (Korge et al., 2018). This intriguing aspartate residue in HEAT H8 (Zhang and Chook, 2012; Niu observation might be related to the fact that PER1 is et al., 2012). The reduction of interaction with Trn1 upon translocated into the nucleus by several karyopherins, but phosphorylation is thereby clearly in accordance with would definitely deserve further examination. structural data, since phosphorylation would cause steric hindrance and electrostatic repulsion between the PY motif 3.1.3 Masking/Unmasking Functional NLS By Folding/ and its binding site on Trn1 (Darovic et al., 2015). Finally, Unfolding Transitions modulation of Trn1-cargo interaction upon phosphorylation In an intriguing example, modulation of Trn1-cargo interaction has also been reported in the case of hnRNP A1 (Allemand would involve the folding/unfolding of a zinc-finger (ZnF). In the et al., 2005). Upon osmotic stress, hnRNP A1 is relocalized from TIS11 family of proteins, a cryptic PY-NLS is masked within a the nucleus to the cytoplasm. This cytoplasmic relocalization is small ZnF domain, the folding of which competes with Trn1- dependent on the phosphorylation of several serine residues mediated nuclear import (Twyffels et al., 2013). It is proposed present at the C-terminus next to the ‘M9’ PY-NLS of hnRNP that TIS11 proteins could be in equilibrium between two states, A1 (Allemand et al., 2005). Importantly, the one in which the ZnF is firmly folded, and one in which it is hyperphosphorylation of hnRNP A1 C-terminal region partially unfolded. This latter state would allow the cryptic PY- reduces its ability to interact with Trn1. The mechanism by NLS to become accessible and thus to mediate nuclear which hyperphosphorylation of serines near the PY-NLS of translocation of the protein (Twyffels et al., 2013). hnRNP A1 leads to impaired binding to Trn1 is unclear, but it has been proposed that these phosphorylations might modulate 3.1.4 RNA-Mediated Regulation of Trn1-Cargo the accessibility of the PY-NLS (Allemand et al., 2005). Interactions Interestingly, in a completely different context, osmotic stress Another example of regulation of nuclear import by Trn1 consists also caused the cytoplasmic translocation of FUS in neurons in the RNA-mediated regulation of ADAR1 import. As presented (Hock et al., 2018). In this case, relocalization of FUS upon above, the non-PY-NLS of ADAR1 is bimodular and formed by osmotic stress does not depend on phosphorylations of the cargo, the N- and C-terminal extensions flanking a dsRBD. This but is caused by a global impairment of Trn1-mediated nuclear molecular organization was shown to act as an RNA-sensing import. As a consequence, several Trn1 cargoes (e.g., EWS, NLS that can be switched on and off, depending on the presence TAF15, and hnRNP A1) also displayed a marked cytoplasmic of dsRNA associated with the dsRBD (Fritz et al., 2009; Barraud shift in neurons (Hock et al., 2018). et al., 2014). The non-PY-NLS of ADAR1 presents two functional and non-overlapping interaction surfaces, namely a functional 3.1.2 Reactive Oxidative Species Regulating dsRNA-binding interface involving the central dsRBD, and a Trn1-Cargo Interactions functional Trn1-binding interface that consists of the N- and As already introduced above with the example of FOXO4, C-terminal modules flanking the folded domain. Even if these reactive oxidative species (ROS) can modulate Trn1-cargo two interfaces are distinct, the non-PY-NLS of ADAR1 cannot interaction, and thereby regulate nuclear import (Putker et al., bind to dsRNA and Trn1 simultaneously, most probably for steric 2013). In this example, accumulation of ROS leads to the reasons, which explains how this non-PY-NLS functions as an formation of a covalent complex between FOXO4 NLS and RNA-sensing NLS (Barraud et al., 2014). Interestingly, RNA- Trn1, which ensures a strong Trn1-cargo interaction and an binding enhances nuclear export of ADAR1 (Fritz et al., 2009), efficient nuclear import (Putker et al., 2013). In another example, which suggests that ADAR1 might leave the nucleus bound ROS also stimulates the interaction of Trn1 with the Parkinson to substrate RNAs. The binding of the non-PY-NLS to protein 7 (PARK7/DJ-1), but the mechanism seems very different Trn1 in the cytoplasm could then help the dissociation of (Björkblom et al., 2014). In this system, H2O2 treatment disrupts ADAR1-bound RNAs and the RNA-sensing NLS would then the DJ-1 dimeric complex, which renders the otherwise masked prevent ADAR1 from carrying RNAs back into the nucleus PY-NLS accessible for Trn1 binding, thereby activating nuclear (Barraud et al., 2014). import of monomeric DJ-1. Here ROS enhances the binding of In another system, RNA has been shown not to impair, but to the DJ-1 PY-NLS to Trn1, but the effect is likely indirect and stimulate Trn1-cargo interaction. A circular RNA named triggered by DJ-1 monomerization (Björkblom et al., 2014). In a circAnks1a, was indeed shown to enhance the interaction last example, it was also shown that ROS stimulates the between the transcription factor YB-1 and Trn1, thus interaction of Trn1 with the circadian clock protein PERIOD promoting the Trn1-mediated translocation of YB-1 into the 1 (PER1) (Korge et al., 2018). Here, H2O2 treatment resulted in a nucleus (Zhang et al., 2019). The mechanism enabling the PY- dose-dependent increase of Trn1-PER1 interaction. It is worth NLS of YB-1 (Mordovkina et al., 2016) to interact more strongly mentioning that Trn1-binding to PER1 is abolished under with Trn1 in presence of RNA has not yet been investigated and reducing conditions, which suggests that a disulfide bond would deserve further studies. Altogether, these examples might be formed between a cysteine residue of PER1 and a illustrate the multiple layers and the different mechanisms that cysteine residue in Trn1, as reported for FOXO4 (Putker can affect Trn1-cargo interaction and regulate nuclear import.

Frontiers in Molecular Biosciences | www.frontiersin.org10 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

FIGURE 4 | Moonlighting functions of Transportin-1. On one hand, Trn1 has been caught in trafficking proteins destined to cilia (Dishinger et al., 2010; Hurd et al., 2011). For instance, Gli, a transcription factor associated with Hedgehog (Hh) signalling is shown to interact with Trn1 via its ciliary localization signal (CLS) (Han et al., 2017). Ran-GTP in the cilia appears to release Gli from Trn1. Upon Hh signalling Gli is converted to an activated form GliA, a transcriptional activator that then translocates to the nucleus to drive transcription (Kim et al., 2009; Han et al., 2017). On the other hand, Trn1 regulates the aggregation of mislocalized RNA-binding proteins (RBPs) in the cytoplasm (Guo et al., 2018; Hock et al., 2018; Hofweber et al., 2018; Qamar et al., 2018; Yoshizawa et al., 2018). Upon stress or when mutated, FUS translocates into the cytoplasm, where it can aggregate and further solidify into pathologic foci. Trn1 recognizes PY-NLS of FUS and directly drives it back into the nucleus (Dormann et al., 2010). Moreover, Trn1 can additionally bind to the low-complexity (LC) and folded region of FUS. This binding competes with FUS–FUS interaction and protein-assembly. Thus, PY-NLS serves as a cytoplasmic signal such that mislocalized nuclear RBPs are specifically chaperoned by Trn1 (Guo et al., 2018; Hock et al., 2018; Hofweber et al., 2018; Qamar et al., 2018; Yoshizawa et al., 2018).

4 MOONLIGHTING FUNCTIONS OF transported to cilia. In addition to a classical NLS-like ciliary TRANSPORTIN-1 localization signal (CLS), which depends upon Importin-β, CLSs have been identified that bind Trn1 to mediate their Besides nuclear trafficking, Trn1 has been shown to conduct a ciliary targeting (Fan et al., 2007; Dishinger et al., 2010; larger symphony of various cellular processes. These include Hurd et al., 2011). ciliary transport, stress granule formation, and virus uncoating Retinitis pigmentosa is an X-linked disorder causing night (Figure 4). In this section, we briefly describe the role of Trn1 in blindness that eventually leads to permanent blindness (Patil these processes. et al., 2011). Mutations in the protein Retinitis pigmentosa 2 (RP2) are responsible for the disorder. Trn1-mediated transport 4.1 Ciliary Transport of RP2 to cilia has been demonstrated (Hurd et al., 2011). The Cilia are protrusions on the cell surface, which help to bridge interaction between RP2 and Trn1 is mediated by two distinct cells to the external environment. Primary cilia are frequently sites that bind Trn1 independently. The first binding site is used for cell motility but can also play a key role in both chemo- located at the N-terminus of RP2 and is similar to an NLS. and mechano-sensing. Further, several signalling pathways The other binding site overlaps with the tubulin folding cofactor depend on proper ciliary function. Disruption of ciliary C (TBCC) domain of RP2 and shows slight similarity to the ‘M9’ function leads to ciliopathies, an emerging disease condition sequence of hnRNP A1. Although both of these sites interact with (Reiter and Leroux, 2017). Until recently, a clear picture of Trn1 independently, only the M9-like sequence is essential for proteins destined to ciliary targeting remained obscure. RP2 ciliary targeting (Hurd et al., 2011). Besides, mutations of the Meanwhile, the similarity between nuclear and ciliary M9-like sequence were shown to abolish ciliary targeting of RP2 transport has been demonstrated for several cases where and were found mutated in several human diseases (Patil et al., cargo proteins interact with nuclear receptors to be 2011). Localization of RP2 to the plasma membrane was shown to

Frontiers in Molecular Biosciences | www.frontiersin.org11 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting be a prerequisite for RP2 and Trn1 interaction. The plasma transport machinery (Cohen et al., 2011; Matreyek and membrane association of RP2 is a direct consequence of Engelman, 2013; Tessier et al., 2019). Recent studies have myristoylation and palmitoylation of residues Gly2 and Cys3. shown a direct role of Trn1 in virus uncoating (Fernandez Primary cilia harbour many factors required for Hedgehog et al., 2019; Miyake et al., 2019; Carlon-Andres et al., 2020; (Hh) signalling (Briscoe and Thérond, 2013). Hh signalling plays Yamauchi, 2020). a central role in development and tissue homoeostasis. Mis- Uncoating of Influenza virus A (IVA) happens in three critical regulation of the Hh signalling pathway results in stages: priming, M1 shell dissociation and virus developmental disorders and cancers. Trn1 has been shown to ribonucleoprotein untangling. Trn1 was found among the regulate Hh signalling by trafficking Gli transcription factor into novel hits in short interfering RNA (siRNA)-based infection cilia, where upon Hh signalling Gli is converted to activate the screenings to identify host proteins associated with viral GliA transcriptional activator that then translocates to the nucleus cytosolic uncoating and nuclear import (Miyake et al., 2019). to drive transcription (Figure 4)(Kim et al., 2009; Han et al., Upon Trn1 depletion by siRNA or short hairpin RNA (shRNA) 2017). Interestingly, Trn1 itself is transcriptionally activated by knockdown, a drastic reduction in the infection rate (66–79%) in GliA (Niewiadomski et al., 2014). Gli proteins exhibit both different cell types was observed. Infection was reversed upon classical NLS and PY-NLS. The PY-like NLS along with the expression of GFP-Trn1 that was insensitive to si or sh RNA. C-terminal residues (aa 874–1,080) is vital for the ciliary Immunofluorescence performed 4 hours post infection localization while the classical NLS is responsible for nuclear demonstrated the majority of the M1-ribonucleoprotein localization (Han et al., 2017). A knockdown of Trn1 diminishes (vRNP) to be present in the cytosol in Trn1 depleted cells ciliary localization of Gli both in the presence and in the absence while M1-vRNP was present in the nucleus in MOCK of Hg signalling. Further, ciliary localization of Gli is reverted on depleted cells (Miyake et al., 2019). Glycine 18 and adjacent transfecting Trn1 highlighting that Trn1 is necessary for Gli residues of M1 was identified to be essential for an interaction ciliary localization (Han et al., 2017). In addition, it was with Trn1. Further, a G18A mutation in M1 was shown to affect shown that Trn1 is required for Hh signalling and is therefore both viral assembly and uncoating, ultimately resulting in a critical for zebrafish embryonic development. drastic reduction in infection (Yamauchi, 2020). The atomic Intra-flagellar transport (IFT) is mainly propelled by Kinesin- structure of G18A showed no major rearrangement when 2 family members (Dishinger et al., 2010; Verhey et al., 2011). compared to wild-type M1, indicating that the mutation most KIF17 is a member of the Kinesin-2 family. The similarity likely affects the interaction with Trn1. between Trn1-mediated nuclear and ciliary trafficking had Capsid protein (CA) multimers encase the RNA genome of been initially suggested from the conservation of common human immunodeficiency virus-1 (HIV-1) (Campbell and Hope, features (Devos et al., 2004), and direct evidence were reported 2015). The capsid is believed to provide a secured micro- few years later (Dishinger et al., 2010). It has been shown that environment for the viral reverse transcriptase to transform Trn1 and RanGTP govern ciliary entry of KIF17 (Dishinger et al., RNA genome to double-stranded DNA and to evade the cell’s 2010). Indeed, the authors have identified NLS-like sequences in innate immune system (Lahaye et al., 2013; Rasaiyaah et al., KIF17: amino acids 767–777 (KRRKR) and 1,016–1,019 (KRKK). 2013). It is largely accepted that timely dissolution of CA is Only mutations in 1,016–1,019 (CLS) of the KIF17 tail domain mandatory for efficient infection but the exact mechanism abolished ciliary targeting and interaction with Trn1, whereas remains unclear. Since HIV-1 uncoating is linked to nuclear mutations in aa 767–772 did not affect ciliary entry. These entry, Trn1 seems to play a critical role in HIV-1 infection findings indicate that Trn1 indeed governs the trafficking of (Fernandez et al., 2019). To test this, the authors knocked KIF17 to cilia. Moreover, the C-terminal tail domain (amino down Trn1, which led to a significant reduction in HIV-1 acids 801–1,028) when fused to a non-ciliary kinesin promotes infection. While Trn1 transfection restored HIV-1 infection, localization to cilia, indicating that this CLS is necessary and implying a direct role of Trn1 in HIV-1 infection. Although sufficient for ciliary transport. Consistently, a mutated CLS fused Trn1 and Trn2 share 83% identity, Trn2 knockdown did not to non-ciliary kinesin did not promote transport into cilia. affect HIV-1 infection, suggesting a specific function of Trn1. Further, the presence of RanGTP in cilia has been Moreover, a significant reduction in infection was seen in Trn1 demonstrated using RanGTP specific antibodies (Fan et al., depleted CD4+ cells, substantiating that Trn1 is a prerequisite for 2011). Also mass spectrometric analyses have documented the early steps of infection (Fernandez et al., 2019). CA and Trn1 presence of Trn1 and RanGTP in the cilium (Liu et al., 2007; could be co-immunoprecipitated in HIV-1- infected cells, Ishikawa et al., 2012; Narita et al., 2012). indicating interaction of Trn1 with CA. Further, Trn1 colocalization with CA in the cytosol and at the nuclear pore 4.2 Virus Uncoating was shown. CA exhibits a hydrophobic patch containing a glycine Understanding the molecular mechanisms of host-virus residue at position 89 (Fernandez et al., 2019). Such a glycine interactions is of great medical interest. Virus uncoating is one residue was shown to be critical for interaction of hnRNP A1 PY- of the critical steps in virus infection which involves the timely NLS with Trn1 (Lee et al., 2006). Consistently, the glycine at release of the viral genome from its shell/capsid (Yamauchi and position 89 (G89) is conserved throughout HIV-1 subtypes Greber, 2016; Helenius, 2018). The stimulus and mechanistic (Kuiken et al., 2003) and G89 in conjunction with the proline details of virus uncoating remain largely concealed. Virus at position 90 (P90) was shown to bind the peptidylprolyl infection in eukaryotic cells often depends on the nuclear isomerase CypA (Gamble et al., 1996; Yoo et al., 1997). A

Frontiers in Molecular Biosciences | www.frontiersin.org12 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

G89V mutation drastically reduced the infection rate when This leads to FUS cytoplasmic retention and subsequent compared to a P90A mutation, suggesting a dependency of disease manifestation (Dormann et al., 2010). However, G89 for the interaction with Trn1. Further, the dependency of recent studies showed an additional chaperoning function Trn1-CA interaction on G89 was shown in vitro by surface of Trn1 that shapes phase separation of FUS and other FET plasmon resonance (Fernandez et al., 2019). The authors proteins. The PY-NLS anchors Trn1 to mislocalized cargos demonstrated the HIV-1 CA uncoating dependency on Trn1 and promotes additional weak binding to low complexity and by an elegant fate-of-capsid assay on sedimentation gradients. folded regions which compete with self-aggregation of FET Soluble CA, representing fully uncoated capsid was only present proteins (Figure 4)(Guo et al., 2018; Hofweber et al., 2018; in control HeLa cytosolic fractions and not in Trn1 knockdown Qamar et al., 2018; Yoshizawa et al., 2018). This chaperoning lysates. Purified recombinant Trn1 was also shown to cause function seems to be independent of Trn1-mediated nuclear structural damage to the assembled capsid/nucleocapsid import, as import mutants of Trn1 (Chook et al., 2002) still (CANC) by atomic force microscopy. Additionally, W730 of prevent and reverse FUS fibrillization. In addition, Trn1 can Trn1 was shown to be critical for the interaction with CA and specifically extract its cargos from SGs without affecting SG- uncoating (Fernandez et al., 2019). Molecular docking formation per se (Guo et al., 2018). Thus, in this case, the NLS simulations revealed that Trn1 may have other points of might serve as a signal that ensures that nuclear cargos are contact in addition to G89 with CA hexamers and it may chaperoned and disaggregated when they are trapped in the induce strong hindrance leading to uncoating. Moreover, Trn1 cytoplasm (Figure 4)(Guo et al., 2018; Hofweber et al., 2018; depletion significantly reduced nuclear accumulation of both CA Qamar et al., 2018; Yoshizawa et al., 2018). Similar and viral DNA. While CA and viral pre-integration complex mechanisms have been proposed for the Trn1-mediated DNA (PIC DNA) accumulated in the nucleus on Trn1 disaggregation of the amyloid fibrils formed by hnRNP A1 expression, suggesting that Trn1 traffics both capsid and LC domain, thus conferring a protective activity against PIC DNA. hnRNPA1-drivenALSandMSP(Sun et al., 2020). Chaperoning is presumably a common feature of 4.3 Regulation of Stress Granule Formation importins, given that Impα/Impβ could prevent TDP43 As mentioned previously, nuclear import of numerous RNA- fibrillization in vitro (Guo et al., 2018). Moreover, Impα/ binding proteins (RBPs) is controlled by Trn1, including the FET Impβ inhibits phase separation of FUS if fused to a protein family (FUS, EWS, TAF15) and hnRNP A1/A2 (Lee et al., classical NLS (Yoshizawa et al., 2018). Importin-mediated 2006). Upon stress response or when mutated, these disaggregation might be similar to the transition of the predominantly nuclear proteins can translocate to the nuclear pore complex, where importins break weak cytoplasm and accumulate in stress granules (SGs). SGs can hydrophobic interactions between FG-Nups (Schmidt and further mature into pathogenic inclusions that are typical for a Görlich, 2016). Corresponding to the interaction with group of fatal neurodegenerative disorders including nucleoporins, Trn1 interacts with tyrosines in the FUS low- amyotrophic lateral sclerosis (ALS), frontotemporal dementia complexity domain. Moreover, Trn1 contains highly acidic (FTD), and multisystem proteinopathy (MSP) (Dormann surfaces and loops that possibly interact with the basic RGG et al., 2010; Harrison and Shorter, 2017; Neumann et al., regions of FUS (Yoshizawa et al., 2018). Thus, Trn1 and 2011). Recent studies show, that Trn1 not only acts as a presumably importins in general, might serve as transport receptor but presumably serves as a powerful cytoplasmic chaperones, that regulate the dynamics of cytoplasmic chaperone for mislocalized RBPs. Thus, Trn1 phase separation and thereby the content of stress granules. regulates SGs formation and their further fibrillization (Figure 4)(Guo et al., 2018; Hock et al., 2018; Hofweber 4.1.2 Chaperoning Modulation By Arginine et al., 2018; Qamar et al., 2018; Yoshizawa et al., 2018; Sun Methylation and RNA-Binding et al., 2020). Arginine methylation can modulate not only direct recognition and binding to FUS-NLS as mentioned before (Dormann et al., 4.1.1 Trn1 is a Chaperone of Mislocalized 2012), but also chaperoning of FUS by Trn1. FUS-FTD is usually FET-Proteins not linked to FUS mutations although its manifestation is FET proteins are involved in many steps of RNA metabolism analogous to ALS. Immunohistochemistry, however, revealed such as transcription and splicing (Lagier-Tourenne et al., 2010). that the FUS-FTD inclusions contain hypomethylated Moreover, they possess additional cytoplasmic function e.g., FUS arginines, hence suggesting a different pathomolecular modulates the axonal mRNA transport and local translation mechanism that leads to fibril formation in FUS-FTDs (Fujii and Takumi, 2005; López-Erauskin et al., 2020). FET (Dormann et al., 2012). Cation-π interaction between proteins share a common domain-architecture: An N-terminal hypomethylated arginines in RGG and tyrosines in LC Prion-like (PrLD) or low complexity domain (LC), followed by promotes FUS condensation into stable inter-molecular several arginine-glycine-glycine (RGG) regions, an RNA β-sheet-rich hydrogels, that cannot be disassociated by recognition motif (RRM) and a C-terminal PY-NLS (Iko et al., physiological Trn1-level (Qamar et al., 2018). Thus, 2004; Springhower et al., 2020). methylation of arginines in RGG regions tunes the strength of ALS-associated FUS mutations are usually found in the PY- FUS intermolecular interactions that promote phase separation NLS and directly disturb nuclear import mediated by Trn1. (Hofweber et al., 2018; Qamar et al., 2018).

Frontiers in Molecular Biosciences | www.frontiersin.org13 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

In the nucleus, high concentration of RNA saturates binding in these processes and the related molecular mechanisms remain properties of FUS molecules and inhibits phase separation. In largely unknown. As for the recognition of non-PY-NLSs, contrary, the low RNA concentration in the cytoplasm may detailed biochemical and structural studies would for instance promote FUS-RNP assembly (Maharana et al., 2018). RGG greatly help understand how Trn1 interacts with substrates regions bind RNA and Trn1 in a mutually exclusive manner. destined to ciliary trafficking. Thus, RNA replacement by Trn1 might also contribute to FUS Nowadays, Trn1 attracts a lot of attention for its clear chaperoning (Hofweber et al., 2018; Yoshizawa et al., 2018). ALS- protective activity in RNA-binding protein phase separation associated arginine-mutations lead to aberrant RNA binding that and maturation of their inclusions in cells. Overexpression of is static and, on the contrary to wild-type FUS, does not switch Trn1 reverses aberrant phase separation in vitro and in cells into dynamic binding stage and promotes larger droplets (Guo et al., 2018; Hofweber et al., 2018; Yoshizawa et al., formation (Niaki et al., 2020). 2018). Trn1 restores FUS-mRNA targets expression in ALS- derived fibroblast and rescues impaired protein synthesis in axon terminals (Guo et al., 2018; Qamar et al., 2018). Studies 5 CONCLUDING REMARKS in flymodelsshowedanincreaseinlifespan,whenmutated FUS was co-expressed with Trn1 in motor neurons, and The recognition at a molecular level of NLSs by Trn1 is now complete rescue of MSP-linked hnRNPA2 mutation (Guo relatively well understood at least for the PY-NLS family. The et al., 2018). Together, these recent studies indicate that recognition of non-PY-NLSs by Trn1 is however far less Trn1 might have a direct therapeutic utility in fatal understood and would definitely deserve comprehensive neurodegenerative diseases. biochemical, structural and cellular studies. These studies focussed on non-PY-NLS recognition by Trn1 would be pivotal to achieve a broad understanding of the mode of AUTHOR CONTRIBUTIONS action of Trn1 and to shed light on this rather unexplored area of Trn1-cargo recognition. It is worth noting that in the All authors listed have made a substantial, direct, and intellectual study of the CIRBP non-PY-NLS, an unexpected role for the H8 contribution to the work and approved it for publication. loop in NLS binding has been unveiled (Bourgeois et al., 2020). The role of the H8 loop has been traditionally restricted to cargo dissociation upon RanGTP binding, and was reported as FUNDING dispensable for NLS binding in early studies (Lee et al., 2006; Imasaki et al., 2007). This new study thereby raises the possibility PB and MJ are supported by a joint ANR-FWF Grant (Nos. ANR- that the Trn1 H8 loop might also be important for binding to 16-CE91-0003 and FWF-I2893). Research at the IBPC is particular NLSs. supported by the CNRS, and the LABEX Dynamo (ANR-11- Although the implication of Trn1 in ciliary trafficking and LABX-0011). Research in the lab of MJ is supported by FWF virus uncoating has been clearly established, the binding of Trn1 Grant numbers F80-07 and P32678.

REFERENCES Baake, M., Bäuerle, M., Doenecke, D., and Albig, W. (2001). Core histones and linker histones are imported into the nucleus by different pathways. Eur. J. Cell Biol. 80, 669–677. doi:10.1078/0171-9335-00208 Allegretti, M., Zimmerli, C. E., Rantos, V., Wilfling, F., Ronchi, P., Fung, H. K. H, Bader, A. G., and Vogt, P. K. (2005). Inhibition of protein synthesis by Y box- et al. (2020). In-cell architecture of the nuclear pore and snapshots of its binding protein 1 blocks oncogenic cell transformation. Mol. Cell Biol. 25, turnover. Nature 14, 33–39. doi:10.1038/s41586-020-2670-5 2095–2106. doi:10.1128/MCB.25.6.2095-2106.2005 Allemand, E., Guil, S., Myers, M., Moscat, J., Cáceres, J. F., and Krainer, A. R. Banerjee, S., and Barraud, P. (2014). Functions of double-stranded RNA-binding (2005). Regulation of heterogenous nuclear ribonucleoprotein A1 transport by domains in nucleocytoplasmic transport. RNA Biol. 11, 1226–1232. doi:10. phosphorylation in cells stressed by osmotic shock. Proc. Natl. Acad. Sci. U.S.A. 4161/15476286.2014.972856 102, 3605–3610. doi:10.1073/pnas.0409889102 Barraud, P., and Allain, F. H. (2012). ADAR proteins: double-stranded RNA and Andrade, M. A., and Bork, P. (1995). HEAT repeats in the Huntington’s disease Z-DNA binding domains. Curr. Top. Microbiol. Immunol. 353, 35–60. doi:10. protein. Nat. Genet. 11, 115–116. doi:10.1038/ng1095-115 1007/82_2011_145 Andrade, M. A., Petosa, C., O’Donoghue, S. I., Müller, C. W., and Bork, P. (2001). Barraud,P.,andAllain,F.H.(2013).SolutionstructureofthetwoRNA Comparison of ARM and HEAT protein repeats. J. Mol. Biol. 309, 1–18. doi:10. recognition motifs of hnRNP A1 using segmental isotope labeling: how 1006/jmbi.2001.4624 the relative orientation between RRMs influences the nucleic acid Arenas, A., Chen, J., Kuang, L., Barnett, K. R., Kasarskis, E. J., Gal, J., et al. (2020). binding topology. J. Biomol. NMR 55, 119–138. doi:10.1007/s10858- Lysine acetylation regulates the RNA binding, subcellular localization and 012-9696-4 inclusion formation of FUS. Hum. Mol. Genet. 29, 2684–2697. doi:10.1093/ Barraud,P.,Banerjee,S.,Mohamed,W.I.,Jantsch,M.F.,andAllain,F.H. hmg/ddaa159 (2014). A bimodular nuclear localization signal assembled via an extended Arnold, M., Nath, A., Hauber, J., and Kehlenbach, R. H. (2006). Multiple importins double-stranded RNA-binding domain acts as an RNA-sensing signal for function as nuclear transport receptors for the Rev protein of human transportin 1. Proc. Natl. Acad. Sci. U.S.A. 111, E1852–E1861. doi:10.1073/ immunodeficiency virus type 1. J. Biol. Chem. 281, 20883–20990. doi:10.1074/ pnas.1323698111 jbc.M602189200 Beck, M., and Hurt, E. (2017). The nuclear pore complex: understanding its Baade, I., and Kehlenbach, R. H. (2019). The cargo spectrum of nuclear transport function through structural insight. Nat. Rev. Mol. Cell Biol. 18, 73–89. doi:10. receptors. Curr. Opin. Cell Biol. 58, 1–7. doi:10.1016/j.ceb.2018.11.004 1038/nrm.2016.147

Frontiers in Molecular Biosciences | www.frontiersin.org14 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

Bischoff, F. R., Klebe, C., Kretschmer, J., Wittinghofer, A., and Ponstingl, H. (1994). the nuclear pore complex. Mol. Biol. Cell 8, 2379–2390. doi:10.1091/mbc.8. RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Proc. Natl. Acad. 12.2379 Sci. U.S.A. 91, 2587–2591. doi:10.1073/pnas.91.7.2587 Devos, D., Dokudovskaya, S., Alber, F., Williams, R., Chait, B. T., Sali, A., Rout, M. Bischoff, F. R., and Görlich, D. (1997). RanBP1 is crucial for the release of RanGTP P., et al. (2004). Components of coated vesicles and nuclear pore complexes from importin beta-related nuclear transport factors. FEBS Lett. 419, 249–254. share a common molecular architecture. PLoS Biol. 2, e380. doi:10.1371/ doi:10.1016/s0014-5793(97)01467-1 journal.pbio.0020380 Björkblom, B., Maple-Grødem, J., Puno, M. R., Odell, M., Larsen, J. P., and Møller, Dishinger,J.F.,Kee,H.L.,Jenkins,P.M.,Fan,S.,Hurd,T.W.,Hammond,J.W., S. G. (2014). Reactive oxygen species-mediated DJ-1 monomerization et al. (2010). Ciliary entry of the kinesin-2 motor KIF17 is regulated by modulates intracellular trafficking involving karyopherin β2. Mol. Cell Biol. importin-beta2 and RanGTP. Nat. Cell Biol. 12, 703–710. doi:10.1038/ 34, 3024–3040. doi:10.1128/MCB.00286-14 ncb2073 Blackwell, J S., Jr, Wilkinson, S. T., Mosammaparast, N., and Pemberton, L. F. Dormann,D.,Rodde,R.,Edbauer,D.,Bentmann,E.,Fischer,I.,Hruscha,A., (2007). Mutational analysis of H3 and H4 N termini reveals distinct roles et al. (2010). ALS-associated fused in sarcoma (FUS) mutations disrupt in nuclear import. J. Biol. Chem. 282, 20142–20150. doi:10.1074/jbc. Transportin-mediated nuclear import. EMBO J. 29, 2841–2857. doi:10. M701989200 1038/emboj.2010.143 Bonifaci, N., Moroianu, J., Radu, A., and Blobel, G. (1997). Karyopherin beta2 Dormann, D., Madl, T., Valori, C. F., Bentmann, E., Tahirovic, S., Abou-Ajram, C., mediates nuclear import of a mRNA binding protein. Proc. Natl. Acad. Sci. et al. (2012). Arginine methylation next to the PY-NLS modulates Transportin U.S.A. 94, 5055–5060. doi:10.1073/pnas.94.10.5055 binding and nuclear import of FUS. EMBO J. 31, 4258–4275. doi:10.1038/ Bourgeois, B., Hutten, S., Gottschalk, B., Hofweber, M., Richter, G., Sternat, J., et al. emboj.2012.261 (2020). Nonclassical nuclear localization signals mediate nuclear import of Eckmann, C. R., Neunteufl, A., Pfaffstetter, L., and Jantsch, M. F. (2001). The CIRBP. Proc Natl Acad Sci U S A 117, 8503–8514. doi:10.1073/pnas.1918944117 human but not the Xenopus RNA-editing enzyme ADAR1 has an atypical Briscoe, J., and Thérond, P. P. (2013). The mechanisms of Hedgehog signalling and nuclear localization signal and displays the characteristics of a shuttling protein. its roles in development and disease. Nat. Rev. Mol. Cell Biol. 14, 416–429. Mol. Biol. Cell 12, 1911–1924. doi:10.1091/mbc.12.7.1911 doi:10.1038/nrm3598 Fan, X. C., and Steitz, J. A. (1998). HNS, a nuclear-cytoplasmic shuttling Campbell, E. M., and Hope, T. J. (2015). HIV-1 capsid: the multifaceted key sequence in HuR. Proc. Natl. Acad. Sci. U.S.A. 95, 15293. doi:10.1073/ player in HIV-1 infection. Nat. Rev. Microbiol. 13, 471–483. doi:10.1038/ pnas.95.26.15293 nrmicro3503 Fan, S., Fogg, V., Wang, Q., Chen, X. W., Liu, C. J., and Margolis, B. (2007). A novel Cansizoglu, A. E., and Chook, Y. M. (2007). Conformational heterogeneity of Crumbs3 isoform regulates cell division and ciliogenesis via importin beta karyopherin beta2 is segmental. Structure 15, 1431–1441. doi:10.1016/j.str. interactions. J. Cell Biol. 178, 387–398. doi:10.1083/jcb.200609096 2007.09.009 Fan, S., Whiteman, E. L., Hurd, T. W., McIntyre, J. C., Dishinger, J. F., Liu, C. J., Cansizoglu, A. E., Lee, B. J., Zhang, Z. C., Fontoura, B. M., and Chook, Y. M. (2007). et al. (2011). Induction of Ran GTP drives ciliogenesis. Mol. Biol. Cell 22, Structure-based design of a pathway-specific nuclear import inhibitor. Nat. 4539–4548. doi:10.1091/mbc.E11-03-0267 Struct. Mol. Biol. 14, 452–454. doi:10.1038/nsmb1229 Fernandez, J., Machado, A. K., Lyonnais, S., Chamontin, C., Gärtner, K., Léger, T., Carlon-Andres, I., Lagadec, F., Pied, N., Rayne, F., Lafon, M. E., Kehlenbach, R. H., et al. (2019). Transportin-1 binds to the HIV-1 capsid via a nuclear localization et al. (2020). Nup358 and Transportin 1 cooperate in adenoviral genome signal and triggers uncoating. Nat. Microbiol. 4, 1840–1850. doi:10.1038/ import. J. Virol. 94, 33. doi:10.1128/JVI.00164-20 s41564-019-0575-6 Chook, Y. M., and Blobel, G. (1999). Structure of the nuclear transport Floer, M., and Blobel, G. (1996). The nuclear transport factor karyopherin beta complex karyopherin-beta2-Ran x GppNHp. Nature 399, 230–237. binds stoichiometrically to Ran-GTP and inhibits the Ran GTPase doi:10.1038/20375 activating protein. J. Biol. Chem. 271, 5313–5316. doi:10.1074/jbc.271. Chook, Y. M., Jung, A., Rosen, M. K., and Blobel, G. (2002). Uncoupling Kapbeta2 10.5313 substrate dissociation and ran binding. Biochemistry 41, 6955–6966. doi:10. Fridell, R. A., Truant, R., Thorne, L., Benson, R. E., and Cullen, B. R. (1997). 1021/bi012122p Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to Chook, Y. M., and Süel, K. E. (2011). Nuclear import by karyopherin-βs: karyopherin-beta. J. Cell Sci. 110 (Pt 11), 1325–1331. recognition and inhibition. Biochim. Biophys. Acta 1813, 1593–606. doi:10. Fried, H., and Kutay, U. (2003). Nucleocytoplasmic transport: taking an 1016/j.bbamcr.2010.10.014 inventory. Cell Mol. Life Sci. 60, 1659–1688. doi:10.1007/s00018-003- Cingolani,G.,Petosa,C.,Weis,K.,andMüller,C.W.(1999).Structureof 3070-3 importin-beta bound to the IBB domain of importin-alpha. Nature 399, Fritz, J., Strehblow, A., Taschner, A., Schopoff, S., Pasierbek, P., and Jantsch, M. 221–229. doi:10.1038/20367 F. (2009). RNA-regulated interaction of transportin-1 and exportin-5 with Cohen, S., Au, S., and Panté, N. (2011). How viruses access the nucleus. Biochim. the double-stranded RNA-binding domain regulates nucleocytoplasmic Biophys. Acta 1813, 1634–45. doi:10.1016/j.bbamcr.2010.12.009 shuttlingofADAR1.Mol. Cell Biol. 29, 1487–1497. doi:10.1128/MCB. Conti, E., and Izaurralde, E. (2001). Nucleocytoplasmic transport enters the 01519-08 atomic age. Curr. Opin. Cell Biol. 13, 310–319. doi:10.1016/s0955-0674(00) Fronz, K., Güttinger, S., Burkert, K., Kühn, U., Stöhr, N., Schierhorn, A., et al. 00213-1 (2011). Arginine methylation of the nuclear poly(a) binding protein weakens Conti, E., Müller, C. W., and Stewart, M. (2006). Karyopherin flexibility in the interaction with its nuclear import receptor, transportin. J. Biol. Chem. 286, nucleocytoplasmic transport. Curr Opin Struct Biol 16, 237–244. doi:10. 32986–32994. doi:10.1074/jbc.M111.273912 1016/j.sbi.2006.03.010 Fu, X., Liang, C., Li, F., Wang, L., Wu, X., Lu, A., et al. (2018). The rules and Cook, A., Bono, F., Jinek, M., and Conti, E. (2007). Structural biology of functions of nucleocytoplasmic shuttling proteins. Int. J. Mol. Sci. 19, 129. nucleocytoplasmic transport. Annu. Rev. Biochem. 76, 647–671. doi:10.1146/ doi:10.3390/ijms19051445 annurev.biochem.76.052705.161529 Fujii, R., and Takumi, T. (2005). TLS facilitates transport of mRNA encoding an Cook, A. G., and Conti, E. (2010). Nuclear export complexes in the frame. Curr. actin-stabilizing protein to dendritic spines. J. Cell Sci. 118, 5755–5765. doi:10. Opin. Struct. Biol. 20, 247–252. doi:10.1016/j.sbi.2010.01.012 1242/jcs.02692 Darovic, S., Prpar Mihevc, S., Zupunski,ˇ V., Guncar,ˇ G., Stalekar,ˇ M., Lee, Y. B., Fukuhara, N., Fernandez, E., Ebert, J., Conti, E., and Svergun, D. (2004). et al. (2015). Phosphorylation of C-terminal tyrosine residue 526 in FUS Conformational variability of nucleo-cytoplasmic transport factors. J. Biol. impairs its nuclear import. J. Cell Sci. 128, 4151–4159. doi:10.1242/jcs.176602 Chem. 279, 2176–2181. doi:10.1074/jbc.M309112200 Dasso, M. (2002). The Ran GTPase: theme and variations. Curr Biol 12, Gamble, T. R., Vajdos, F. F., Yoo, S., Worthylake, D. K., Houseweart, M., Sundquist, R502–R508. doi:10.1016/s0960-9822(02)00970-3 W. I., et al. (1996). Crystal structure of human cyclophilin A bound to the Delphin, C., Guan, T., Melchior, F., and Gerace, L. (1997). RanGTP targets p97 amino-terminal domain of HIV-1 capsid. Cell 87, 1285–1294. doi:10.1016/ to RanBP2, a filamentous protein localized at the cytoplasmic periphery of s0092-8674(00)81823-1

Frontiers in Molecular Biosciences | www.frontiersin.org15 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

Görlich, D., and Kutay, U. (1999). Transport between the cell nucleus and the Kimura, M., Morinaka, Y., Imai, K., Kose, S., Horton, P., and Imamoto, N. (2017). cytoplasm. Annu. Rev. Cell Dev. Biol 15, 607–660. doi:10.1146/annurev.cellbio. Extensive cargo identification reveals distinct biological roles of the 12 importin 15.1.607 pathways. Elife 6, 121. doi:10.7554/eLife.21184 Guo, L., Kim, H. J., Wang, H., Monaghan, J., Freyermuth, F., Sung, J. C., et al. Klebe, C., Bischoff, F. R., Ponstingl, H., and Wittinghofer, A. (1995). (2018). Nuclear-import receptors reverse aberrant phase transitions of RNA- Interaction of the nuclear GTP-binding protein Ran with its regulatory binding proteins with prion-like domains. Cell 173, 677–e20. doi:10.1016/j.cell. proteins RCC1 and RanGAP1. Biochemistry 34, 639–647. doi:10.1021/ 2018.03.002 bi00002a031 Güttinger, S., Mühlhäusser, P., Koller-Eichhorn, R., Brennecke, J., and Kutay, U. Klucevsek, K., Daley, J., Darshan, M. S., Bordeaux, J., and Moroianu, J. (2006). (2004). Transportin2 functions as importin and mediates nuclear import of Nuclear import strategies of high-risk HPV18 L2 minor capsid protein. HuR. Proc. Natl. Acad. Sci. U.S.A. 101, 2918–2923. doi:10.1073/pnas. Virology 352, 200–208. doi:10.1016/j.virol.2006.04.007 0400342101 Korge, S., Maier, B., Brüning, F., Ehrhardt, L., Korte, T., Mann, M., et al. (2018). Hampoelz, B., Andres-Pons, A., Kastritis, P., and Beck, M. (2019). Structure and The non-classical nuclear import carrier Transportin 1 modulates circadian assembly of the nuclear pore complex. Annu. Rev. Biophys. 48, 515–536. doi:10. rhythms through its effect on PER1 nuclear localization. PLoS Genet. 14, 1146/annurev-biophys-052118-115308 e1007189. doi:10.1371/journal.pgen.1007189 Han, Y., Xiong, Y., Shi, X., Wu, J., Zhao, Y., and Jiang, J. (2017). Regulation of Gli Kuiken, C., Korber, B., and Shafer, R. W. (2003). HIV sequence databases. AIDS ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-β2 Rev. 5, 52–61. doi:10.2172/1566101 nuclear import system. PLoS Biol. 15, e2002063. doi:10.1371/journal.pbio. Lagier-Tourenne, C., Polymenidou, M., and Cleveland, D. W. (2010). TDP-43 and 2002063 FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum. Harrison, A. F., and Shorter, J. (2017). RNA-binding proteins with prion-like Mol. Genet. 19, R46–R64. doi:10.1093/hmg/ddq137 domains in health and disease. Biochem. J. 474, 1417–1438. doi:10.1042/ Lahaye, X., Satoh, T., Gentili, M., Cerboni, S., Conrad, C., Hurbain, I., et al. (2013). BCJ20160499 The capsids of HIV-1 and HIV-2 determine immune detection of the viral Helenius, A. (2018). Virus entry: looking back and moving forward. J. Mol. Biol. cDNA by the innate sensor cGAS in dendritic cells. Immunity 39, 1132–1142. 430, 1853–1862. doi:10.1016/j.jmb.2018.03.034 doi:10.1016/j.immuni.2013.11.002 Hock, E. M., Maniecka, Z., Hruska-Plochan, M., Reber, S., Laferrière, F., Le Roux, L. G., and Moroianu, J. (2003). Nuclear entry of high-risk human Sahadevan, M. K. S., et al. (2018). Hypertonic stress causes cytoplasmic papillomavirus type 16 E6 oncoprotein occurs via several pathways. J. Virol. translocation of neuronal, but not astrocytic, FUS due to impaired 77, 2330–2337. doi:10.1128/jvi.77.4.2330-2337.2003 transportin function. Cell Rep. 24, 987–e7. doi:10.1016/j.celrep.2018. Lee, S. J., Matsuura, Y., Liu, S. M., and Stewart, M. (2005). Structural basis for 06.094 nuclear import complex dissociation by RanGTP. Nature 435, 693–696. doi:10. Hofweber, M., Hutten, S., Bourgeois, B., Spreitzer, E., Niedner-Boblenz, A., 1038/nature03578 Schifferer, M., et al. (2018). Phase separation of FUS is suppressed by its Lee, B. J., Cansizoglu, A. E., Süel, K. E., Louis, T. H., Zhang, Z., and Chook, Y. M. nuclear import receptor and arginine methylation. Cell 173, 706–e13. doi:10. (2006). Rules for nuclear localization sequence recognition by karyopherin beta 1016/j.cell.2018.03.004 2. Cell 126, 543–558. doi:10.1016/j.cell.2006.05.049 Huber, F. M., and Hoelz, A. (2017). Molecular basis for protection of ribosomal Leemann-Zakaryan, R. P., Pahlich, S., Grossenbacher, D., and Gehring, H. (2011). protein L4 from cellular degradation. Nat. Commun. 8, 14354. doi:10.1038/ Tyrosine Phosphorylation in the C-terminal nuclear localization and retention ncomms14354 signal (C-NLS) of the EWS protein. Sarcoma 2011, 218483. doi:10.1155/2011/ Hülsmann, B. B., Labokha, A. A., and Görlich, D. (2012). The permeability of 218483 reconstituted nuclear pores provides direct evidence for the selective phase Liu, Q., Tan, G., Levenkova, N., Li, T., Pugh, E. N., Jr, Rux, J. J., et al. (2007). The model. Cell 150, 738–751. doi:10.1016/j.cell.2012.07.019 proteome of the mouse photoreceptor sensory cilium complex. Mol. Cell Hurd, T. W., Fan, S., and Margolis, B. L. (2011). Localization of retinitis pigmentosa Proteomics 6, 1299–1317. doi:10.1074/mcp.M700054-MCP200 2 to cilia is regulated by Importin beta2. J. Cell Sci. 124, 718–726. doi:10.1242/ López-Erauskin,J.,Tadokoro,T.,Baughn,M.W.,Myers,B.,McAlonis- jcs.070839 Downes, M., Chillon-Marinas, C., et al. (2020). ALS/FTD-linked Iko, Y., Kodama, T. S., Kasai, N., Oyama, T., Morita, E. H., Muto, T., et al. (2004). mutation in FUS suppresses intra-axonal protein synthesis and drives Domain architectures and characterization of an RNA-binding protein, TLS. disease without nuclear loss-of-function of FUS. Neuron 106, 354. doi:10. J. Biol. Chem. 279, 44834–44840. doi:10.1074/jbc.M408552200 1016/j.neuron.2020.04.006 Imasaki,T.,Shimizu,T.,Hashimoto,H.,Hidaka,Y.,Kose,S.,Imamoto,N., Lukong, K. E., Larocque, D., Tyner, A. L., and Richard, S. (2005). Tyrosine et al. (2007). Structural basis for substrate recognition and dissociation by phosphorylation of sam68 by breast tumor kinase regulates intranuclear human transportin 1. Mol. Cell 28, 57–67. doi:10.1016/j.molcel.2007. localization and cell cycle progression. J. Biol. Chem. 280, 38639–38647. 08.006 doi:10.1074/jbc.M505802200 Ishikawa, H., Thompson, J., Yates, J. R., 3rd, and Marshall, W. F. (2012). Proteomic Mackmull, M. T., Klaus, B., Heinze, I., Chokkalingam, M., Beyer, A., Russell, R. B., analysis of mammalian primary cilia. Curr. Biol. 22, 414–419. doi:10.1016/j.cub. et al. (2017). Landscape of nuclear transport receptor cargo specificity. Mol. 2012.01.031 Syst. Biol. 13, 962. doi:10.15252/msb.20177608 Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I. W., and Görlich, D. (1997). The Maharana, S., Wang, J., Papadopoulos, D. K., Richter, D., Pozniakovsky, A., asymmetric distribution of the constituents of the Ran system is essential for Poser, I., et al. (2018). RNA buffers the phase separation behavior of prion- transport into and out of the nucleus. EMBO J. 16, 6535–6547. doi:10.1093/ like RNA binding proteins. Science 360, 918–921. doi:10.1126/science. emboj/16.21.6535 aar7366 Jäkel, S., and Görlich, D. (1998). Importin beta, transportin, RanBP5 and RanBP7 Matreyek, K. A., and Engelman, A. (2013). Viral and cellular requirements for the mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J. 17, nuclear entry of retroviral preintegration nucleoprotein complexes. Viruses 5, 4491–4502. doi:10.1093/emboj/17.15.4491 2483–2511. doi:10.3390/v5102483 Kim, J., Kato, M., and Beachy, P. A. (2009). Gli2 trafficking links Hedgehog- Michael, W. M., Choi, M., and Dreyfuss, G. (1995). A nuclear export signal in dependent activation of Smoothened in the primary cilium to transcriptional hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export activation in the nucleus. Proc. Natl. Acad. Sci. U.S.A. 106, 21666–21667. doi:10. pathway. Cell 83, 415–422. doi:10.1016/0092-8674(95)90119-1 1073/pnas.0912180106 Miyake, Y., Keusch, J. J., Decamps, L., Ho-Xuan, H., Iketani, S., Gut, H., et al. Kimura, M., Kose, S., Okumura, N., Imai, K., Furuta, M., Sakiyama, N., et al. (2019). Influenza virus uses transportin 1 for vRNP debundling during cell (2013). Identification of cargo proteins specificforthenucleocytoplasmic entry. Nat. Microbiol. 4, 578–586. doi:10.1038/s41564-018-0332-2 transport carrier transportin by combination of an in vitro transport system Mordovkina,D.A.,Kim,E.R.,Buldakov,I.A.,Sorokin,A.V.,Eliseeva,I.A., and stable isotope labeling by amino acids in cell culture (SILAC)-based Lyabin, D. N., et al. (2016). Transportin-1-dependent YB-1 nuclear import. quantitative proteomics. Mol. Cell Proteomics 12, 145–157. doi:10.1074/mcp. Biochem. Biophys. Res. Commun. 480, 629–634. doi:10.1016/j.bbrc.2016. M112.019414 10.107

Frontiers in Molecular Biosciences | www.frontiersin.org16 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

Mosammaparast, N., Guo, Y., Shabanowitz, J., Hunt, D. F., and Pemberton, L. F. Siomi, M. C., Eder, P. S., Kataoka, N., Wan, L., Liu, Q., and Dreyfuss, G. (1997). (2002). Pathways mediating the nuclear import of histones H3 and H4 in yeast. Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins. J. Biol. Chem. 277, 862–888. doi:10.1074/jbc.M106845200 J. Cell Biol. 138, 1181–1192. doi:10.1083/jcb.138.6.1181 Mühlhäusser, P., Müller, E. C., Otto, A., and Kutay, U. (2001). Multiple pathways Soniat, M., Sampathkumar, P., Collett, G., Gizzi, A. S., Banu, R. N., Bhosle, R. C., contribute to nuclear import of core histones. EMBO Rep. 2, 690–696. doi:10. et al. (2013). Crystal structure of human Karyopherin β2 bound to the PY-NLS 1093/embo-reports/kve168 of Saccharomyces cerevisiae Nab2. J. Struct. Funct. Genom. 14, 31–35. doi:10. Narita, K., Kozuka-Hata, H., Nonami, Y., Ao-Kondo, H., Suzuki, T., Nakamura, H., 1007/s10969-013-9150-1 et al. (2012). Proteomic analysis of multiple primary cilia reveals a novel mode Soniat, M., and Chook, Y. M. (2016). Karyopherin-β2 recognition of a PY-NLS of ciliary development in mammals. Biol. Open 1, 815–825. doi:10.1242/bio. variant that lacks the proline–tyrosine motif. Structure 24, 1802–1809. doi:10. 20121081 1016/j.str.2016.07.018 Neumann, M., Bentmann, E., Dormann, D., Jawaid, A., DeJesus-Hernandez, Soniat, M., Cagatay,˘ T., and Chook, Y. M. (2016). Recognition Elements in the M.,Ansorge,O.,etal.(2011).FETproteinsTAF15andEWSareselective Histone H3 and H4 Tails for Seven Different Importins. J. Biol. Chem. 291, markers that distinguish FTLD with FUS pathology from amyotrophic 21171–21183. doi:10.1074/jbc.M116.730218 lateral sclerosis with FUS mutations. Brain 134, 2595–609. doi:10.1093/ Springhower,C.E.,Rosen,M.K.,andChook,Y.M.(2020).Karyopherinsand brain/awr201 condensates. Curr. Opin. Cell Biol. 64, 112–123. doi:10.1016/j.ceb.2020. Niaki, A. G., Sarkar, J., Cai, X., Rhine, K., Vidaurre, V., Guy, B., et al. (2020). Loss of 04.003 dynamic RNA interaction and aberrant phase separation induced by two Stewart, M., Kent, H. M., and McCoy, A. J. (1998). The structure of the Q69L distinct types of ALS/FTD-linked FUS mutations. Mol. Cell 77, 82–e4. mutant of GDP-Ran shows a major conformational change in the switch II loop doi:10.1016/j.molcel.2019.09.022 that accounts for its failure to bind nuclear transport factor 2 (NTF2). J. Mol. Niewiadomski, P., Kong, J,. H., Ahrends, R., Ma, Y., Humke, E. W., Khan, S., et al. Biol. 284, 1517–1527. doi:10.1006/jmbi.1998.2204 (2014). Gli protein activity is controlled by multisite phosphorylation in Strehblow, A., Hallegger, M., and Jantsch, M. F. (2002). Nucleocytoplasmic vertebrate Hedgehog signaling. Cell Rep. 6, 168–181. doi:10.1016/j.celrep. distribution of human RNA-editing enzyme ADAR1 is modulated by 2013.12.003 double-stranded RNA-binding domains, a leucine-rich export signal, and a Niu, C., Zhang, J., Gao, F., Yang, L., Jia, M., Zhu, H., et al. (2012). FUS-NLS/ putative dimerization domain. Mol. Biol. Cell 13, 3822–3835. doi:10.1091/mbc. Transportin 1 complex structure provides insights into the nuclear targeting E02-03-0161 mechanism of FUS and the implications in ALS. PLoS One 7, e47056. doi:10. Suárez-Calvet, M., Neumann, M., Arzberger, T., Abou-Ajram, C., Funk, E., 1371/journal.pone.0047056 Hartmann, H., et al. (2016). Monomethylated and unmethylated FUS Patil, S. B., Hurd, T. W., Ghosh, A. K., Murga-Zamalloa, C. A., and Khanna, H. exhibit increased binding to Transportin and distinguish FTLD-FUS (2011). Functional analysis of retinitis pigmentosa 2 (RP2) protein reveals from ALS-FUS. Acta Neuropathol. 131, 587–604. doi:10.1007/s00401- variable pathogenic potential of disease-associated missense variants. PLoS One 016-1544-2 6, e21379. doi:10.1371/journal.pone.0021379 Süel, K. E., Gu, H., and Chook, Y. M. (2008). Modular organization and Pollard, V. W., Michael, W. M., Nakielny, S., Siomi, M. C., Wang, F., and Dreyfuss, combinatorial energetics of proline–tyrosine nuclear localization signals. G. (1996). A novel receptor-mediated nuclear protein import pathway. Cell 86, PLoS Biol. 6, e137. doi:10.1371/journal.pbio.0060137 985–994. doi:10.1016/s0092-8674(00)80173-7 Sun, Y., Zhao, K., Xia, W, Feng, G., Gu, J., Ma, Y., et al. (2020). The nuclear Putker, M., Madl, T., Vos, H. R., de Ruiter, H., Visscher, M., van den Berg, M. C., localization sequence mediates hnRNPA1 amyloid fibril formation revealed et al. (2013). Redox-dependent control of FOXO/DAF-16 by transportin-1. Mol by cryoEM structure. Nat. Commun. 11, 6349. doi:10.1038/s41467-020- Cell 49, 730–742. doi:10.1016/j.molcel.2012.12.014 20227-8 Qamar, S., Wang, G., Randle, S. J., Ruggeri, F. S., Varela, J. A., Lin, J. Q., et al. Suzuki, M., Iijima, M., Nishimura, A., Tomozoe, Y., Kamei, D., and Yamada, M. (2018). FUS phase separation is modulated by a molecular chaperone and (2005). Two separate regions essential for nuclear import of the hnRNP D methylation of arginine cation-π interactions. Cell 173, 720–e15. doi:10.1016/j. nucleocytoplasmic shuttling sequence. FEBS J. 272, 3975–3987. doi:10.1111/j. cell.2018.03.056 1742-4658.2005.04820.x Rasaiyaah, J., Tan, C. P., Fletcher, A. J., Price, A. J., Blondeau, C., Hilditch, L., et al. Tai, L. R., Chou, C. W., Lee, I. F., Kirby, R., and Lin, A. (2013). The quantitative (2013). HIV-1 evades innate immune recognition through specific cofactor assessment of the role played by basic amino acid clusters in the nuclear uptake recruitment. Nature 503, 402–405. doi:10.1038/nature12769 of human ribosomal protein L7. Exp. Cell Res. 319, 367–375. doi:10.1016/j. Rebane, A., Aab, A., and Steitz, J. A. (2004). Transportins 1 and 2 are redundant yexcr.2012.12.007 nuclear import factors for hnRNP A1 and HuR. RNA 10, 590–599. doi:10.1261/ Tessier, T. M., Dodge, M. J., Prusinkiewicz, M. A., and Mymryk, J. S. (2019). Viral rna.5224304 appropriation: laying claim to host nuclear transport machinery Cell 8, 13. Reiter, J. F., and Leroux, M. R. (2017). Genes and molecular pathways doi:10.3390/cells8060559 underpinning ciliopathies. Nat. Rev. Mol. Cell Biol. 18, 533–547. doi:10. Tran, E. J., and Wente, S. R. (2006). Dynamic nuclear pore complexes: life on the 1038/nrm.2017.60 edge. Cell 125, 1041–1053. doi:10.1016/j.cell.2006.05.027 Rout, M. P., Aitchison, J. D., Magnasco, M. O., and Chait, B. T. (2003). Virtual Truant, R., Kang, Y., and Cullen, B. R. (1999). The human tap nuclear RNA export gating and nuclear transport: the hole picture. Trends Cell Biol 13, 622–688. factor contains a novel transportin-dependent nuclear localization signal that doi:10.1016/j.tcb.2003.10.007 lacks nuclear export signal function. J. Biol. Chem. 274, 32167–32171. doi:10. Scheffzek, K., Klebe, C., Fritz-Wolf, K., Kabsch, W, and Wittinghofer, A. (1995). 1074/jbc.274.45.32167 Crystal structure of the nuclear Ras-related protein Ran in its GDP-bound form. Twyffels, L., Wauquier, C., Soin, R., Decaestecker, C., Gueydan, C., and Kruys, Nature 374, 378–381. doi:10.1038/374378a0 V. (2013). A masked PY-NLS in Drosophila TIS11 and its mammalian Schmidt, H. B., and Görlich, D. (2016). Transport selectivity of nuclear pores, phase homolog tristetraprolin. PLoS One 8, e71686. doi:10.1371/journal.pone. separation, and membraneless organelles. Trends Biochem. Sci. 41, 46–61. 0071686 doi:10.1016/j.tibs.2015.11.001 Twyffels, L., Gueydan, C., and Kruys, V. (2014). Transportin-1 and Transportin-2: Seewald, M. J., Körner, C., Wittinghofer, A., and Vetter, I. R. (2002). RanGAP protein nuclear import and beyond. FEBS Lett. 588, 1857–1868. doi:10.1016/j. mediates GTP hydrolysis without an arginine finger. Nature 415, 662–666. febslet.2014.04.023 doi:10.1038/415662a Verhey, K. J., Dishinger, J., and Kee, H. L. (2011). Kinesin motors and primary cilia. Shamsher, M. K., Ploski, J., and Radu, A. (2002). Karyopherin beta 2B participates Biochem. Soc. Trans. 39, 1120–1125. doi:10.1042/BST0391120 in mRNA export from the nucleus. Proc. Natl. Acad. Sci. U.S.A. 99, Vetter, I. R., Nowak, C., Nishimoto, T., Kuhlmann, J., and Wittinghofer, A. (1999). 14195–14196. doi:10.1073/pnas.212518199 Structure of a Ran-binding domain complexed with Ran bound to a GTP Siomi, H., and Dreyfuss, G. (1995). A nuclear localization domain in the hnRNP A1 analogue: implications for nuclear transport. Nature 398, 39–46. doi:10.1038/ protein. J. Cell Biol. 129, 551–560. doi:10.1083/jcb.129.3.551 17969

Frontiers in Molecular Biosciences | www.frontiersin.org17 February 2021 | Volume 8 | Article 638149 Mboukou et al. Trn1: Nuclear Import and Moonlighting

Wang, X., Xu, X., Zhu, S., Xiao, Z., Ma, Z., Li, Y., et al. (2012). Molecular dynamics Yoshizawa,T.,Ali,R.,Jiou,J.,Fung,HYJ,Burke,K.A.,Kim,S.J.,etal. simulation of conformational heterogeneity in transportin 1. Proteins 80, (2018). Nuclear import receptor inhibits phase separation of FUS through 382–397. doi:10.1002/prot.23193 binding to multiple sites. Cell 173, 693–e22. doi:10.1016/j.cell.2018. Weighardt, F., Biamonti, G., and Riva, S. (1995). Nucleo-cytoplasmic distribution 03.003 of human hnRNP proteins: a search for the targeting domains in hnRNP A1. Zhang, Z. C., and Chook, Y. M. (2012). Structural and energetic basis of ALS- J. Cell Sci. 108 (Pt 2), 545–555. causing mutations in the atypical proline–tyrosine nuclear localization signal of Weis, K. (2003). Regulating access to the genome: nucleocytoplasmic transport the Fused in Sarcoma protein (FUS). Proc. Natl. Acad. Sci. U.S.A. 109, throughout the cell cycle. Cell 112, 441–451. doi:10.1016/s0092-8674(03) 12017–12021. doi:10.1073/pnas.1207247109 00082-5 Zhang, S. B., Lin, S. Y., Liu, M., Liu, C. C., Ding, H. H., Sun, Y., et al. (2019). Weis, K. (2007). The nuclear pore complex: oily spaghetti or gummy bear?. Cell CircAnks1a in the spinal cord regulates hypersensitivity in a rodent model 130, 405–407. doi:10.1016/j.cell.2007.07.029 of neuropathic pain. Nat. Commun. 10, 4119. doi:10.1038/s41467-019- Wente, S. R., and Rout, M. P. (2010). The nuclear pore complex and nuclear 12049-0 transport. Cold Spring Harb. Perspect. Biol. 2, a000562. doi:10.1101/cshperspect. a000562 Conflict of Interest: The authors declare that the research was conducted in the Xu, D., Farmer, A., and Chook, Y. M. (2010). Recognition of nuclear targeting absence of any commercial or financial relationships that could be construed as a signals by Karyopherin-β proteins. Curr. Opin. Struct. Biol. 20, 782–790. doi:10. potential conflict of interest. 1016/j.sbi.2010.09.008 Yamauchi, Y., and Greber, U. F. (2016). Principles of virus uncoating: cues and the Copyright © 2021 Mboukou, Rajendra, Kleinova, Tisné, Jantsch and Barraud. This snooker ball. Traffic 17, 569–592. doi:10.1111/tra.12387 is an open-access article distributed under the terms of the Creative Commons Yamauchi, Y. (2020). Influenza A virus uncoating. Adv. Virus Res. 106, 1–38. Attribution License (CC BY). The use, distribution or reproduction in other forums is doi:10.1016/bs.aivir.2020.01.001 permitted, provided the original author(s) and the copyright owner(s) are credited Yoo, S., Myszka, D. G., Yeh, C., McMurray, M., Hill, C. P., and Sundquist, W. I. and that the original publication in this journal is cited, in accordance with accepted (1997). Molecular recognition in the HIV-1 capsid/cyclophilin A complex. academic practice. No use, distribution or reproduction is permitted which does not J. Mol. Biol. 269, 780–95. doi:10.1006/jmbi.1997.1051 comply with these terms.

Frontiers in Molecular Biosciences | www.frontiersin.org18 February 2021 | Volume 8 | Article 638149